Methods for multiple direct label probe detection of multiple chromosomes or regions thereof by in situ hybridization

ABSTRACT

Direct label probe compositions which stain DNA of a preselected single chromosome or region of a chromosome of a multi-chromosomal genome are provided that comprise mixed DNA segments which are covalently bound to fluorophore groups through linking groups. The mixed DNA segments are derived from the DNA present in the preselected chromosome or chromosome region. These probe compositions can be used concurrently or sequentially with other probe compositions.

RELATED APPLICATIONS

This application is a CON of Ser. No. 09/110,562 filed Jul. 6, 1998, nowU.S. Pat. No. 6,277,569; which is a DIV of Ser. No. 08/781,682 filedJan. 10, 1997, now U.S. Pat. No. 5,776,688; which is a CON of Ser. No.08/476,694 filed Jun. 7, 1995, now U.S. Pat. No. 5,663,319; which is aDIV of Ser. No. 08/222,167 filed Apr. 4, 1994 now U.S. Pat. No.5,491,224; which is a CON of Ser. No. 07/762,913 filed Sep. 19, 1991,now abandoned; which is a CIP of Ser. No. 07/585,876, filed Sep. 20,1990, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the detection and identification ofchromosomes or regions of chromosomes by hybridization of a multiplicityof different chromosome-specific probes. In particular, this inventionrelates to in situ hybridization of these chromosome specific probes tothe target chromosome. The present invention also relates to thedetection of chromosomes or regions of chromosomes using fluorescentlylabeled reagents.

Background of the Invention

Probes containing DNA sequences which are complementary to target DNAsequences of specific individual whole chromosomes or regions ofchromosomes of a multi-chromosomal genome are known (see, for example,Pinkel et al. in “Fluorescence in situ hybridization with humanchromosome-specific libraries: Detection of trisomy 21 andtranslocations of chromosome 4” at Proc. Nat'l Acad. Sci. USA85:9138-9142, December 1988; Manuelidis in “Chromosomal Localization ofComplex and Simple Repeated Human DNA's” Chromosoma 66:23-32, 1978).

The vast majority of prior art probes prepared from such sequences wereindirect label probes and so required post-hybridization processing.Thus, for example, such probes were derivatized with biotin, and,following the hybridization procedure, steps were pursued to build asandwich-like structure of fluorescein-labeled avidin and biotinylatedanti-avidin antibodies. In contrast, the direct label probes of thisinvention require only one probe penetration step of a slide mountedspecimen during an in situ hybridization procedure.

Prior art methods for labeling such prior art chromosome-specificcomplementary DNA sequences present difficulties in controlling thenumber of label moieties attached to individual sequences.

Improved probe compositions comprised of (a) fluorophore labels whichare easily and accurately directly detected, and (b) DNA segments whichare complementary to specific chromosomal DNA segments would be veryuseful as chromosome specific stains in in situ hybridization assays.The present invention provides both such probes and efficient, reliablemethods for their preparation and use.

SUMMARY OF THE INVENTION

The present invention provides: (1)probe compositions for the in situdetection of a chromosome or a region of a chromosome, (2)methods forthe preparation of such probe compositions, and (3)methods for the useof such probe compositions for the in situ detection of a chromosome ora region of a chromosome.

This invention provides probe compositions for in situ detection of apreselected chromosome or region of a chromosome comprising multiple DNAsegments complementary to different portions of the chromosome orchromosome region to be detected where, in the probe compositions, DNAsegments include multiple fluorescent labels covalently linked thereto.

The invention includes a probe composition that contains unhybridizedDNA segments which are essentially complementary to DNA base sequencesexisting in different portions of the chromosome or chromosome region tobe detected and which contain a plurality of cytosine bases (i.e.,deoxycytidine nucleotides). A number of the cytosine bases have afluorescent label covalently bonded thereto. The number of fluorescentlylabeled cytosine bases is sufficient to generate a detectablefluorescent signal while the individual so labeled DNA segmentsessentially retain their specific complementary binding (hybridizing)properties with respect to the chromosome or chromosome region to bedetected.

The invention also includes a method for making a probe composition forin situ detection of a particular preselected chromosome or a region ofsuch chromosome comprising:

(a) disrupting (that is, fragmenting) DNA complementary to thechromosome, or the region of the chromosome, into fragments,

(b) transaminating the DNA fragments, and

(c) covalently linking a fluorescent dye to the transaminated DNAfragments.

More specifically, the invention includes a method for preparing a probecomposition for in situ detection of a preselected chromosome or regionof a chromosome comprising:

(a) transaminating with a linking group a number of deoxycytidinenucleotides contained in unhybridized DNA base sequences or segmentsthat are essentially representative of complementary base targetsequences existing in the chromosome or chromosome region to bedetected; and

(b) covalently bonding a fluorescent label to at least a portion of thetransaminated deoxycytidine bases, the portion of deoxycytidine baseshaving fluorescent labels covalently bonded thereto being sufficient togenerate a detectable fluorescent signal while essentially retaining thespecific complementary binding properties of the transaminated baseswith respect to the chromosome or the chromosome region to be detected.

In addition, the invention provides a method for in situ detection of apreselected chromosome or a region of the chromosome comprising:

(a) adding an excess of blocking DNA to an inventive probe compositionpreferably under hybridizing conditions to bond with nonspecific bindingDNA in the probe composition, thereby forming a blocked probecomposition,

(b) contacting the blocked probe composition under hybridizingconditions with the chromosome or the chromosome region to be detected,and

(c) detecting the binding of the blocked probe composition to thechromosome or the chromosome region to be detected by fluorescenttechniques.

It is an additional object of the present invention to provide probecompositions that are directly labeled with fluorescent dyes. The use ofsuch directly labeled probe compositions avoids the need for detailedand lengthy post-hybridization procedures as required by the indirectlylabeled prior art probe compositions using, for example, biotin labels,avidin, and biotinylated antiavidin antibodies. The inventive probecompositions permit the user to proceed immediately from thehybridization step to final washing and visualization, thereby reducingthe necessary amount of assay time and labor. The number of reagentsrequired to perform the assay is also reduced, resulting in enhancedsimplicity of use and manufacturing.

It is a specific object of the present invention to provide a probecomposition for the in situ detection of a preselected chromosome or apreselected region of a chromosome wherein the probe compositioncontains multiple DNA segments that are complementary to differentportions of the chromosome or the chromosome region to be detected. Themultiple DNA segments in the probe composition include multiplefluorescent labels that are covalently linked to the DNA segments. Thesefluorescent labels permit the DNA segments that hybridize with thechromosome or the chromosome region to be detected using fluorescenttechniques. In a preferred embodiment, the fluorescent labels arecovalently linked to a number of the deoxycytidine bases in the DNAsegments through linking groups. The number of deoxycytidine baseshaving fluorescent labels is sufficient to generate a detectablefluorescent signal, while at the same time the specific bindingproperties of the labeled DNA segments are essentially retained withrespect to the chromosome or the chromosome region to be detected.

It is also an object of this invention to detect preselected multiplechromosomes or regions of chromosomes. This is accomplished by labelingone library of DNA segments specific for one chromosome or chromosomeregion with one fluorescent label and labeling another library of DNAsegments specific for another chromosome or chromosome region withanother fluorescent label, such that the fluorescent labels canindependently be detected by fluorescent techniques. DNA segments ofeach chromosome or chromosome region of interest are thus so labeled.Thus, combinations of such resulting probe compositions of thisinvention can be used to detect two or more chromosomes or regions ofchromosomes.

This invention also comprises a method for detecting a plurality ofpreselected chromosomes or regions of chromosomes, by providing a probecomposition of such labeled specifically binding DNA segments for eachchromosome or region of chromosome of the preselected plurality. Theprobe composition specific to each chromosome or chromosome region islabeled with a different fluorescent label such that each fluorescentlabel can be detected in the presence of the others. These probecompositions of labeled DNA are then contacted either in admixture or insuccession under hybridizing conditions with the chromosomes or theregions of the chromosomes to be detected. Hybridization is detected bythe presence or absence of the particular fluorescent signal generatedby each of the labeled DNA segments which has hybridized with theparticular selected chromosome or the particular selected chromosomeregion.

The detection of multiple chromosomes or chromosomal regions isconsiderably more difficult using an indirect labeling technique sincedifferent indirect binding partners (e.g. biotin/avidin orantibody/antigen) are required for each chromosome or region to beidentified. For example, detection of two different chromosomes wouldrequire biotin/avidin and a specific antibody/antigen binding pair, inaddition to two different detectable labels, such as fluorophores. Theindirect label technique, therefore, requires six label components ascompared to only two label components required by the direct labeltechnique. The detection of three events requires the addition of athird different set of antibody/antigen partners and a third fluorophore(nine label components total compared to three components in the directlabel technique). It is much more difficult to find and adapt anadditional pair of binding partners than it is to find an additionalfluorophore. A large number of fluorophores are commercially availablein reactive forms amenable to labeling transaminated DNA.

It is an additional specific object of the present invention to providepreferred methods for making probe compositions for the in situdetection of a preselected chromosome or preselected region of achromosome. The first step of a preferred inventive method is to disruptplasmid DNA derived from a phage chromosomal library into fragments.These DNA fragments are transaminated with a linking group andfluorescent dyes are then covalently linked to the transaminated DNAfragments. In a preferred embodiment of the present invention, thenumber of transaminated deoxycytidine nucleotides to which fluorescentlabels are covalently bonded is sufficient to generate a detectablefluorescent signal while at the same time the specific bindingproperties of the DNA segments are essentially retained with respect tothe chromosome or chromosome region to be detected.

It is a further specific object of the present invention to providepreferred methods for the in situ detection of a preselected chromosomeor preselected region of a chromosome. In general, the preferred methodsare carried out by contacting a probe composition of the presentinvention with the preselected chromosome or chromosome region to bedetected under hybridizing conditions. The presence or absence of thefluorescent signal generated by a probe composition that has beenhybridized with the chromosome or the chromosome region of interest isthen detected. In a preferred embodiment, an excess of blocking DNA isadded to the probe composition preferably under hybridizing conditions,thereby forming a blocked probe composition. This blocking DNA bindswith nonspecific binding DNA in the probe composition. The blocked probecomposition is then contacted with the chromosome or chromosome regionof interest under hybridizing conditions.

Further objects and preferred embodiments of the present invention arediscussed in the following description of the preferred embodiments andclaims.

DETAILED DESCRIPTION (A) Definitions

The term “sequence” refers to a chain or interconnected series of DNAnucleotides.

The term “fragment”, “segment” or “DNA segment” indicates generally onlya portion of a larger DNA polynucleotide or sequence such as occurs inone chromosome or one region of a chromosome. A polynucleotide, forexample, can be broken up, or fragmented into, a plurality of segmentsor fragments. As is known, a chromosome characteristically containsregions which have DNA sequences that contain DNA repeated segments. Theterm “repeated” has reference to the fact that a particular DNA segmentoccurs a plurality (i.e., at least two) of times as the same DNAsequence, or plurality of DNA sequences. Individual DNA segment sizeand/or DNA repeated segment size can vary greatly. For example, in thecase of the human genome, each DNA repeated segment is now believed tobe typically in the approximate size range of about 5 to about 3,000 bp.Illustratively, a single chromosome alphoid DNA sequence may incorporateat least about five different DNA repeated segments.

The term “genome” designates or denotes the complete, single-copy set ofgenetic instructions for an organism as coded into DNA of the organism.In the practice of the present invention, the particular genome underconsideration is typically multi-chromosomal so that such DNA iscellularly distributed among a plurality of individual chromosomes(which number, for example, in humans 22 pairs plus a gender associatedXX pair or an XY pair).

In the practice of this invention, the genome involved in any giveninstance is preferably from a primate, and the DNA sequences of apreselected chromosome from such a genome contain DNA repeated segmentsthat are inclusive of either alphoid DNA or are associated with thecentromere of the preselected chromosome. As used herein, the term“alphoid” or “alpha satellite” in reference to DNA has reference to thecomplex family of tandemly repeated DNA segments found in primategenomes. Long tandem arrays of alpha satellite DNA based on a monomerrepeat length of about 171 base pairs are located principally at thecentromeres of primate chromosomes.

The term “chromosome” refers to a heredity-bearing gene carrier of aliving cell which is derived from chromatin and which comprises DNA andprotein components (especially histones). The conventionalinternationally recognized individual human genome chromosome numberingidentification system is employed herein. The size of an individualchromosome can vary from one type to another with a givenmulti-chromosomal genome and from one genome to another. In the case ofthe (preferred) human genome, the entire DNA mass of a given chromosomeis usually greater than about 100,000,000 bp. For example, the size ofthe entire human genome is about 3×10⁹ bp. The largest chromosome,chromosome no. 1, contains about 2.4×10⁸ bp while the smallestchromosome, chromosome no. 22, contains about 5.3×10⁷ bp (Yunis, J. J.Science 191:1268-1270 (1976), and Kavenoff, R., et al. Cold SpringHarbor Symposia on Quantitative Biology 38:1-8 (1973)).

The term “region” indicates a portion of one chromosome which containsDNA repeated segments that are preferably alphoid or associated with thecentromere. The actual physical size or extent of such an individualregion can vary greatly. An exact quantification of such a region cannotnow be made for all possible regions. Usually, a region is at leastlarge enough to include at least one DNA sequence that (a) incorporatesa plurality of copies of at least one DNA repeated segment and that (b)is identifiable and preferably enumeratable optically by fluoroscopicmicroscopic examination after formation of fluorophore labeled hybridsin such region following an in situ hybridization procedure with adirect label probe or probe composition. Presently available informationsuggests that a region may contain more than a single such DNA sequencewith each such DNA sequence containing one or more DNA repeatedsegments.

The term “region” is typically and characteristically a chromosomefragment which comprises less DNA mass or size than the entire DNA massor size of a given chromosome. As is known, not all the DNA of a givenchromosome or chromosome region is arranged as DNA sequences comprisedof or containing DNA repeated segments. A region, for example, can havea size which encompasses about 2×10⁶ to about 40×10⁶ DNA bp. which sizeregion encompasses, for example, centromeres of the human chromosomes.Such a size is thus a substantial fraction of the size of a single humanchromosome. Such a region size is presently preferred as a region sizein the practice of this invention although larger and smaller regionsizes can be used. A centromeric region of even a small human chromosomeis a microscopically visible large portion of the chromosome, and aregion comprising DNA repeated segments (not alphoid or centromeric) onthe Y chromosome occupies the bulk of the chromosome and ismicroscopically visible. In general, the term “region” is not definitiveof a particular one (or more) genes because a “region” does not takeinto specific account the particular coding segments (exons) of anindividual gene. Rather, a “region” as used herein in reference to achromosome is unique to a given chromosome by reason of the particularcombination of DNA segments therein for present probe compositionformation and use purposes.

The term “centromere” refers to a heterochromatic region of theeucaryotic chromosome which is the chromosomal site of attachment of thekinetochore. The centromere divides just before replicated chromosomesseparate, and so such holds together the paired chromatids.

The term “gene” designates or denotes a DNA sequence along a chromosomethat codes for a functional product (either RNA or its translationproduct, a polypeptide). A gene contains a coding region and includesregions preceding and following the coding region (termed respectively“leader” and “trailer”). The coding region is comprised of a pluralityof coding segments (“exons”) and intervening sequences (“introns”)between individual coding segments.

The term “probe” or “probe composition” refers to a polynucleotide, or amixture of polynucleotides, such as DNA sequence(s) or DNA segment(s)which has (or have) been chemically combined (i.e., associated) withindividual label-containing moieties. Each such polynucleotide of aprobe is typically single stranded at the time of hybridization to atarget.

The term “label” or “label containing moiety” refers in a general senseto a moiety, such as a radioactive isotope or group containing same, andnonisotopic labels, such as enzymes, biotin, avidin, streptavidin,digoxygenin, luminescent agents, dyes, haptens, and the like.Luminescent agents, depending upon the source of exciting energy, can beclassified as radioluminescent, chemiluminescent, bioluminescent, andphotoluminescent (including fluorescent and phosphorescent).

Probe compositions of this invention contain DNA segments that arechemically bound to label-containing moieties. Each label-containingmoiety contains at least one fluorophore (fluorescent) group, and eachlabel-containing moiety is derived from a monofunctional reactivesubstituent-containing, and also fluorophore substituent containing,starting fluorescent compound, as hereinbelow more particularlydescribed.

The term “direct label probe” (or “direct label probe composition”)designates or denotes a nucleic acid probe whose label after hybridformation with a target is detectable without further reactiveprocessing of hybrid. Probe compositions of this invention are of thedirect label type.

The term “indirect label probe” (or “indirect label probe composition”)designates or denotes a nucleic acid probe whose label after hybridformation with a target must be further reacted in subsequent processingwith one or more reagents to associate therewith one or more moietiesthat finally result in a detectable entity.

The term “target”, “DNA target” or “DNA target region” refers to onenucleotide sequence which occurs at a specific chromosomal location.Each such sequence or portion is typically and preferably at leastpartially single stranded (i.e. denatured) at the time of hybridization.When the target nucleotide sequences are located only in a single regionor fraction of a given chromosome, the term “target region” is sometimesapplied. Targets for hybridization can be derived from specimens whichinclude but are not limited to chromosomes or regions of chromosomes innormal, diseased or malignant human or other animal or plant cells,either interphase or at any state of meiosis or mitosis, and eitherextracted or derived from living or postmortem tissues, organs orfluids; germinal cells including sperm and egg cells, seeds, pollen orzygotes, embryos, chorionic or amniotic cells, or cells from any othergerminating body; cells grown in vitro, from either long-term orshort-term culture, and either normal, immortalized or transformed;inter- or intraspecific hybrids of different types of cells ordifferentiation states of these cells; individual chromosomes orportions of chromosomes, or translocated, deleted or other damagedchromosomes, isolated by any of a number of means known to those withskill in the art, including libraries of such chromosomes cloned andpropagated in prokaryotic or other cloning vectors, or amplified invitro by means well known to those with skill; or any forensic material,including but not limited to semen, blood, hair or other samples.

The term “hybrid” refers to the product of a hybridization procedurebetween a probe and a target. Typically, a hybrid is a molecule thatincludes a double stranded, helically configured portion comprised ofcomplementarily paired single stranded molecules, such as two DNAmolecules, one of which is a target DNA nucleotide sequence, and theother of which is the labeled DNA nucleotide sequence of a probe.

The term “stain”, “selective stain”, “selectively stained” or equivalentrefers generally to a localized area color achieved by a stainingprocedure or the like which color takes effect on a selected group ofcomponents (or constituents) of a cytological or histologicalpreparation. Typically, such colored components are to undergo amicroscopic examination, or the like. Commonly another or othersupplementary or background color (i.e., stain) may be involved, such asa so-called counterstain which takes effect on all components of alarger class within which a selectively stained group of componentsfalls. The main purpose of a stain is to enhance or make possibleidentification of components during such an examination. A probecomposition of this invention under hybridizing conditions produceshybrids which in effect stain a target chromosome or target chromosomalregion with a fluorophore group.

The term “fluorescent” (and equivalent terms) has general reference tothe property of a substance (such as a fluorophore) to produce lightwhile it is being acted upon by radiant energy, such as ultravioletlight or x-rays.

The term “fluorescent compound” or “fluorophore group” generally refersto an organic moiety. A fluorescent compound is capable of reacting, anda fluorophore group may have already reacted, with a linking group. Afluorescent compound may include an organic chelator which binds aluminescent inorganic ion such as a rare earth like terbium, europium,ruthenium, or the like.

The term “linking compound” or “linking group” as used herein generallyrefers to a hydrocarbonaceous moiety. A linking compound is capable ofreacting, and a linking group may have already reacted, with anucleotide sequence (or nucleotide segment). A linking compound is alsocapable of reacting, and a linking group may have already reacted, witha fluorophore compound.

The term “in situ” means that the chromosomes are exposed from the cellnucleus without substantial disruption or relocation of the chromosomeswith respect to each other and with the chromosomes being accessible tofluorescently labeled DNA probes.

The term “denaturation” or “denature” has reference to the at leastpartially complete conversion of a polynucleotide from a multi-stranded(or double-stranded) state to a single stranded state. The presence ofan agent or agents which in effect lowers the temperature required fordenaturation and for subsequent hybridizing conditions between probe (orprobe composition) and target is generally desirable, and a presentlymost preferred such agent is formamide. Using, for example, about 50:50volume ratio mixture of water and formamide, an illustrative temperaturefor thermal denaturation is in the range of about 70 to about 80 degreesC. applied for times that are illustratively in the range of about 1 toabout 10 minutes.

The term “in situ hybridization” has reference to hybridization of aprobe to a target that exists within a cytological or histologicalpreparation or specimen. As a result of an in situ hybridizationprocedure, hybrids are produced between a probe and a target. This term“in situ hybridization” may be inclusive of denaturation and may also beinclusive of a hybrid or probe detection procedure which is practicedafter in situ hybridization of a probe to a target. A specimen can beadhered as a layer upon a slide surface, and a specimen can, forexample, comprise or contain individual chromosomes or chromosomeregions which have been treated to maintain their morphology under, forexample, denaturing conditions, or conditions such as typically existduring a flow cytometric analysis in a probe detection procedure. Theterm “in situ hybridization” may include use of a counterstain. In thecase of the inventive fluorophore labeled probes or probe compositions,the detection method can involve fluorescence microscopy, flowcytometry, or the like.

The term “hybridizing conditions” has general reference to thecombinations of conditions that are employable in a given hybridizationprocedure to produce hybrids, such conditions typically involvingcontrolled temperature, liquid phase, and contacting between a probe (orprobe composition) and a target. Conveniently and preferably, at leastone denaturation step precedes a step wherein a probe or probecomposition is contacted with a target. Alternatively, a probe can becontacted with a specimen comprising a DNA target region and bothsubjected to denaturing conditions together as described by Bhatt et al.in Nucleic Acids Research 16: 3951-3961. Using, for example, about a50:50 volume ratio mixture of water and formamide, an illustrativetemperature for contacting and hybridization between probe (or probecomposition) and target is in the range of about 35 to about 55° C.applied for a time that is illustratively in the range of about 1 toabout 18 hours. Other hybridizing conditions can be employed. The ratioof numbers of probes to number of target sequences or segments can varywidely, but generally, the higher this ratio, the higher the probabilityof hybrid formation under hybridizing conditions within limits.

The term “complete”, “completely”, “substantially complete” or“substantially completely” refers to the capacity of a direct labelprobe composition of this invention to hybridize with a target so thatthe target body or bodies is/are highlighted and identifiable (by afluorescence microscope, a flow cytometer, or the like) afterhybridization therewith to an extent at least sufficient to show (i.e.,stain or identify) the target's full extent (morphology). Thus,variations in fluorescence coloration intensity may sometimes be present(i.e., observed) in an individual hybridized chromosomal target body,but the target body as a whole is substantially highlighted.

The term “lower” refers to an individual compound, group or radicalmeans that such compound, group or radical contains less than 6 carbonatoms.

The term “paint probe” or “painting probe”, (or “paint probecomposition” or “painting probe composition”) refers to a probe or probecomposition, such as a probe composition of this invention, which isadapted to hybridize under hybridizing conditions with a target whichcomprises one predetermined (i.e., preselected) chromosome of amulti-chromosomal genome. If only a fractional part of such onechromosome happens to be present in a specimen undergoing such ahybridization with such a probe composition, then such fractional partso hybridizes and is identified. Typically, one paint probe of thisinvention can be admixed with a second so as to make possible thesimultaneous staining and detection of two predetermined chromosomes.

The term “clone”, “cloning” or equivalent refers to the process whereina particular nucleotide segment or sequence is inserted into anappropriate vector, the vector is then transported into a host cell, andthe vector within the host cell is then caused to reproduce itself in aculturing process, thereby producing numerous copies of each vector andthe respective nucleotide sequence that it carries. Cloning results inthe formation of a colony or clone (i.e., group) of identical host cellswherein each contains one or more copies of a vector incorporating aparticular nucleotide segment or sequence. The nucleotide segment orsequence is now said to be “cloned”, and the product nucleotide segmentsor sequences can be called “clones”.

The term “blocking DNA” or “blocking DNA composition” refers to a DNAwhich has the capacity, under hybridizing conditions, to hybridize withnonspecific binding DNA present in a probe. A blocking DNA compositionis comprised of a mixture of DNA segments that are derived from,include, and are preferably representative of, the total genomic DNA ofa multi-chromosomal genome that is under consideration and whichincorporates a target. Such segments can, for example, be prepared byfragmenting (as taught herein) DNA sequences comprising orrepresentative of such total genomic DNA, and such DNA segments soprepared are complementary to DNA segmental portions occurringthroughout the chromosomes (including the regions) of this genome, suchsegments can also be prepared, for example, from a total genomic DNA, byother procedures, such as by a procedure involving the procedural stepsof denaturing partially reannealing or re-hybridizing, and treating withenzymes, thereby to reduce the quantity of non-repeated segmentstherein. Blocking DNA is at the time of use with a probe compositionpreferably in the form of segments having average sizes in the range ofabout 150 to about 600 base pairs.

The term “library” is used herein in its conventional sense to refer toa set of cloned DNA fragments which together represent an entire genomeor a specified fragment thereof, such as a single chromosome. Variouslibraries are known to the prior art and are available from variousrepositories, and techniques for genome and genome fragment preparation,and for cloning libraries therefrom, are well known. A presentprocedural preferences is to fragment a selected one chromosome that wasseparated by flow sorting or the like. Fragmentation prior to cloning ispreferably achieved by digestion with restriction endonucleases or thelike. This procedure produces fragment ends which are particularlyamenable to insertion into vectors. However, those skilled in the artwill appreciate that any conventional or convenient technique forfragmentation can be used. The fragments are then conventionally clonedto produce a chromosome library.

The term “blocked probe composition” has reference to a probecomposition of this invention which is in admixture with a blocking DNAcomposition.

The term “diluent DNA” or equivalent refers to DNA which is the same as,or is similar to the DNA that is incorporated into a particular probecomposition of this invention. Diluent DNA, when admixed withtransaminated polynucleotides that constitute an intermediate in themaking of a probe composition of the invention, or when admixed with aproduct probe composition of this invention, functions to dilute thetotal number of labeled DNA segments that are present in a given volumeor weight of a probe composition of this invention.

As those skilled in the art will appreciate, a diluent DNA can alsosometimes function as a blocking DNA, and vice versa.

The term “carrier DNA” refers to DNA which functions to reduce theamount of probe DNA which is inherently lost due to such effects asabsorption of probe DNA to adjacent surface portions, such as thesurface portions of a container vessel wherein a probe is being stored,the surface portions of a glass slide whereon a specimen undergoing insitu hybridization is deposited, or the surface portions of cellulardebris present in a specimen undergoing in situ hybridization, or thelike. Carrier DNA is comprised of DNA derived from an unrelated genomicspecies, such as salmon sperm in admixture with DNA segments derivedfrom the human genome. A carrier DNA may be optionally added to ahybridization solution that incorporates a probe of this invention.

The term “nonspecific binding DNA” refers to DNA which is complementaryto DNA segments of a probe and which DNA occurs in at least one otherposition in a genome, which other position is outside of a selectedchromosomal target region within that genome. An example of nonspecificbinding DNA comprises a class of DNA repeated segments whose memberscommonly occur in more than one chromosome or chromosome region. Suchcommon repetitive segments tend to hybridize to a greater extent thanother DNA segments that are present in probe composition.

(B) Starting Materials

(1) The Starting Chromosomal DNA

When starting chromosomal DNA is used in the practice of this invention,such is typically and preferably in the form of one or more DNAsequences which taken together contain a multiplicity of DNA segmentsthat individually occur at various locations in and throughout anindividual preselected chromosome of a given multi-chromosomal genomeand that are reasonably representative of DNA occurring in thepreselected chromosome. Although in its naturally occurring state, astarting DNA sequence typically has a size much greater than about onemillion base pairs, at the time of availability for use as a startingmaterial in the practice of this invention, the starting DNA sequencemay already be somewhat fragmented, depending upon such factors as themethods used in separation, isolation and the like. A presentlypreferred genome is the human genome.

For purposes of preparing a given probe composition of this invention,the starting chromosomal DNA sequence(s) can be obtained by varioustechniques. Thus, such can be derived or obtained, for example, from (a)DNA that is separated by flow sorting a plurality of a singlepreselected chromosome of a multi-Chromosomal genome which DNA ispreferably purified from component intracellular material of anorganism; (b) a chromosome library of a preselected chromosome, and (c)an inter species hybrid which incorporates DNA of a preselectedchromosome. A presently preferred starting chromosomal DNA is achromosome library of a preselected chromosome which library has beenprepared by standard methods and is available from traditional sourcesknown to those in the art, such as the American Type Culture Collection(ATCC) or other repositories of human or other cloned genetic material.While a large number of specific chromosome libraries are available fromthe ATCC, representative libraries are shown in Table I:

TABLE I HUMAN CHROMOSOME LIBRARIES Human Human Chromosome ChromosomeLibrary ATCC No. Library ATCC No. 1 57738 13 57757 1 57753 14 57739 157754 14 57706 2 57716 14/15 57707 2 57744 15 57729 3 57717 15 57740 357748 15 57737 3 57751 16 57765 4 57719 16 57730 4 57718 16 57749 457700 16 57758 4 57745 17 57741 5 57720 17 57759 5 57746 18 57742 657721 18 57710 6 57701 19 57731 7 57722 19 57766 7 57755 19 57711 857723 20 57732 8 57707 20 57712 9 57724 21 57743 9 57705 21 57713 1057725 22 57733 10 57736 22 57714 11 57726 X 57750 11 57704 X 57734 1257727 X 57752 12 57736 X 57747 13 57728 Y 57735 13 57705 Y 57715

The ATCC deposits of Table I are available from the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md.

The invention contemplates that such chromosome specific DNA sequencescan also be synthesized in vitro by any one of a number of enzymaticmeans known to those skilled in the art. Specific single chromosomal DNAsequences usable as starting material in the practice of the inventionare isolatable from one or more of these sources by methods which arewell known to those skilled in the art.

Examples of prior art teachings illustrating the preparation of suitablestarting sequences for making whole chromosome paint probes of thisinvention include (but are not limited to):

1. Chromosomes are physically separated, fragmented, and the fragmentspropagated as clones, as in: M. A. Van Dilla, et al. in Bio/Technology4, 537-552 (1986) and Cox, D. R. et al. in Science 250, 245-250 (1990).

2. An entire chromosome is physically scraped from the surface of amicroscope slide, fragmented, and the fragments propagated as clones,using the technique described in: Ludecke, H. J. et al. in Nature 338:348-350 (1989).

3. Single human chromosomes are propagated in rodent cell lines. Thus,the entire DNA content of such a line is fragmented, and the fragmentspropagated as clones. A method for the generation of human,mono-chromosomal hybrid lines is described in: Carlock, L. R. et al. inSomatic Cell Mol. Genet. 12: 163-174 (1986).

4. Sequences from purified single chromosome preparations areenzymatically amplified by utilizing primer oligonucleotidescomplementary to any of a number of abundant, polydisperse repeated DNAsequences which are present at many locations along the chromosome.Purified chromosomal preparations from methods 1, 2 or 3 above aresubjected to amplifications of the type described in: Nelson D. L. etal. in Proc. Natl. Acad. Sci. USA 86: 6686-6690 (1989).

(2) The Starting Regional Chromosomal DNA

When starting regional chromosomal DNA is used in the practice of thisinvention, such is typically and preferably derived either directly orindirectly from one preselected region of one preselected chromosome ofa genome which is preferably multi-chromosomal. Such starting regionalchromosomal DNA is typically in the form of at least one DNA sequence.It is presently preferred that each such sequence or sequencesincorporate a plurality of at least one DNA repeated segment andpreferably a plurality (i.e., at least two) of structurally differingDNA repeated segments. Preferably, such regional DNA sequence is uniquerelative to other regions of the total genome. The starting regional DNAincorporates a multiplicity of DNA segments that occur individually atvarious locations in and throughout the individual preselected region ofone chromosome and that are reasonably representative of DNA occurringin the preselected region. A presently preferred genome is the humangenome.

At the time of involvement in the methodology of this invention, such anindividual DNA regional sequence may be in pieces or fragments, but thesum of the fragments would equal a whole naturally occurring DNAsequence. Such an individual starting DNA sequence can be and preferablyis a cloned or otherwise produced copy of a naturally occurring regionalsequence. Such a starting sequence can have a size which is only afraction of the size of the naturally occurring whole DNA sequence thatis present in the preselected region. However, a given starting regionalchromosomal DNA is reasonably representative of DNA present in thepreselected region.

Various regional chromosomal DNA (and their preparation methods)sequences are known to the prior art and such can be used as startingDNA sequences in the preparation of fluorophore group direct label probecompositions of this invention, and various known techniques can be usedto obtain or prepare such a starting regional chromosomal DNA.

Examples of prior art teachings illustrating methods for obtainingsuitable starting regional DNA sequences that incorporate DNA repeatedsegments include (but are not limited to):

5. Sequences are obtained from cloned pools of DNA enriched in repeatDNAs, as described in; Manuelidis, L., Chromosoma 66: 23-32 (1978), inYang, T. P. et al. in Proc. Natl. Acad. Sci. USA 79: 6593-6597 (1982),and in Moyzis, R. K. et al., in Chromosoma 95: 375-386 (1987).

6. Sequences obtained from purified single chromosome preparations areenzymatically amplified by utilizing primer oligonucleotidescomplementary to conserved portions of chromosome-specific repeated DNAsequences. Purified chromosomal preparations from methods 1, 2 or 3above are subjected to amplifications of the type described in: Koch, J.E., et al. in Chromosoma 98: 259-265 (1989).

7. Sequences obtained from the preparation procedure described incopending application Bittner et al. U.S. Ser. No. 07/762,912, publishedas PCT Patent Application WO 93/0246, on Apr. 1, 1993, filed on evendate herewith. The teachings of this application are incorporated hereinby reference. Such sequences are presently preferred for use in thepresent invention.

Examples of prior art teachings illustrating methods for obtainingstarting DNA sequences which are suitable for making chromosomalregionally specific probes of this invention include (but are notlimited to):

8. Many sequences specific for particular regions of a human chromosomehave been determined. A publicly available compilation of such sequencesis maintained by the National Institute of Health, see: Bilofsky, H. S.et al. in Nucl. Acids Res. 16: 1861-1864 (1988). There are currently5141 individual sequence entries in this compilation. There are6,182,990 base-pairs of sequence information presently provided in theseentries.

9. The physical location of many anonymous DNA segments on the humanchromosome have been described, see: Donis-Keller, H. et al. in Cell 51:319-337 (1987).

10. Known sequences or DNA segments (references 8 and 9 above) are usedas the starting point for obtaining further sequences which are linkedto the available sequence by screening plasmid, cosmid, bacteriophage oryeast artificial chromosome libraries as described in: Wahl, G. M., etal. in Proc. Natl. Acad Sci. USA 84: 2160-2164; Williams, B. G. et al.1979 in J. Virol 29: 555-575 (1982), and Brownstein, B. H. et al. inScience 244: 1348-1351 (1989).

11. Cloned sequences from regions of chromosomes for which there is noknown linkage to an already determined sequence are obtained bymicro-dissection of that region followed by fragmentation, amplificationand cloning, as in; Ludecke, H. J., et al. in Nature 338: 348-350(1989), or by enzymatic amplification of human DNA in interspecificradiation hybrids which contain less than a complete human chromosome,as in Cox, D. R., et al. in Science 250: 245-250 (1990).

For purposes of preparing a direct label probe composition of theinvention, it is presently preferred to employ a starting chromosomalDNA that is representative of the DNA of a selected chromosome orchromosomal region.

In general, a starting chromosomal DNA, or a starting regionalchromosomal DNA, is not required to have DNA segments which have thesame distribution, or occurrence frequency that is characteristic of theDNA segments that naturally (or normally) occurs. By using, for example,a starting chromosomal DNA, or regional chromosomal DNA, wherein theoccurrence or distribution of DNA repeated segments is skewed, thecapacity of a product probe composition to selectively stain targetchromosomal DNA completely is not destroyed, but rather may be altered.The result is that, in a resulting hybridized target in a specimen, thestained chromosomal DNA which is present in a target region may appearto be unevenly stained, but that target region is still substantiallycompletely and selectively stained. The subregions within a stainedtarget displaying greatest coloration intensity when subsequentlyexamined under a fluorescence microscope are believed to correspond totarget chromosomal subregions wherein the relative frequency ofoccurrence of segmental DNA occurring in the probe composition isgreater than at locations where the coloration is weakest. A startingDNA with a skewed distribution of DNA segments compared to a normal ornaturally occurring distribution of DNA segments can be used for test orevaluation purposes, if desired.

The nature, structure and size of individual starting DNA sequences, thenumber of the different sequences utilized, and the like variablesassociated with a starting chromosomal or regional chromosomal DNAcannot be stated in absolute terms because of inherent variations in thegenome from one organism to another, and because the composition of astarting DNA sequence population for a given genome can vary from onesource to another, and even from one batch to another of a particularstarting DNA taken from the same source. However, after fragmentation ofa starting DNA, is achieved, for example, as hereinbelow described, thestarting DNA is preferably converted to a mixture of DNA segments whichmixture comprises segments that are approximately and reasonablyrepresentative of the entire selected chromosome or chromosomal region,and that are preferably distributed throughout such chromosome orregion.

The characteristic complexity of a given starting DNA, however, ispresently considered to be desirable from the standpoint of the presentinvention since such complexity tends to make possible direct labelprobe compositions of the invention which can be prepared from differentstarting DNA from the same selected chromosome or region, yet suchproduct probe compositions behave in a substantially identical manner asregards staining capacity. A starting chromosomal or regional DNAtypically contains about 18 to about 25 mole percent deoxycytidinenucleotides based on the total number of deoxynucleotides presenttherein.

(3) The Starting Linking Compound

A starting linking compound employed in the practice of this inventionis a difunctional organic compound, that is, such contains twosubstituent functional (i.e., reactive) substituents per startinglinking compound molecule.

At least one of such functional substituents per linking compoundmolecule is reactive with deoxycytidine nucleotides in a polynucleotideunder bisulfite catalyzed aqueous transamination conditions (such asprovided herein, for example). Examples of such substituents includealkyl amino (primary and secondary), hydrazido, semicarbazido,thiosemicarbazido, and the like. Amino groups are presently mostpreferred.

When the amino group is secondary, the secondary substituent ispreferably a lower alkyl group, but other non-blocking such secondarysubstituents can be used, if desired.

The second of other of such two functional substituents per linkingcompound molecule is reactive with a third functional substituent whichis itself incorporated into a starting fluorescent compound (as hereindescribed). Such second functional substituent can itself be eitherblocked or unblocked. When the second substituent is unblocked, then itis substantially non-reactive with other substances that are present inthe transamination medium (especially polynucleotides) duringtransamination. When the second substituent is blocked then it issubstantially non-reactive with the other substances that are present inthe transamination medium (especially polynucleotides) duringtransamination.

Examples of suitable unblocked second functional substituent groupinclude amino, carboxyl, phosphate, sulfonate, hydroxyl, hydrazido,semicarbazido, thiosemicarbazido and the like. Presently, most preferredunblocked second functional substituent include amino (primary orsecondary) and carboxyl groups.

The carboxyl group preferably is either in the salt form or in the acidform, but can sometimes be in the ester form. When in the salt form,presently preferred cations are alkali metals, such as sodium andpotassium.

Examples of suitable blocked second functional substituent group includeblocked sulfonate, blocked phosphate, blocked sulfhydryl, and the like.

Examples of suitable blocking substituents include lower alkyl groupssuch as methyl, ethyl, propyl, etc.

The first and the second functional substituents are interconnectedtogether through a linker (or linking) moiety. This linking moiety canhave any convenient structure but such is non-reactive with othersubstances that are present in the transamination medium duringtransamination. A present preference is that the linking moiety be ahydrocarbonaceous divalent group which is acyclic or cyclical and whichcan optionally incorporate other atoms.

The two functional substituents present in such a difunctional linkingcompound can be respective substituents of the linking moiety. Suchsubstituents can be on adjacent carbon atoms relative to each other, orthey can be spaced from one another in a linking compound molecule by aplurality of intervening interconnected atoms (preferably carbon atoms).Preferably these functional groups are in an alpha, omega relationshipto one another (that is, each is at a different opposite end region) ina given linking compound molecule.

Thus, the two functional radicals in a linking compound are each bondedto an organic linking group moiety which is either entirelyhydrocarbonaceous (that is, composed only of carbon and hydrogen atoms),or is comprised of carbon and hydrogen atoms plus at least oneadditional atom or group which contains at least one atom selected fromthe group consisting of oxygen, sulfur, nitrogen, phosphorous, or thelike. Preferably such additional atom(s) are so associated with suchorganic moiety as to be substantially less reactive than either one ofsuch above indicated two functional radicals that are present in a givenstarting linking compound. Hydrocarbonaceous organic moieties that aresaturated aliphatic are presently preferred, and more preferably suchmoiety is a divalent alkylene radical containing from 2 through 12carbon atoms, inclusive. However, if desired, such a saturated aliphaticradical can incorporate either at least one ether group (—O—) or atleast one thio-ether group (—S—), but it is presently more preferredthat only one of such ether or thio ether groups be present. It ispresently preferred that a linking compound incorporates an organicradical that contains at least two and not more than about a total ofabout 20 carbon atoms, although more carbon atoms per molecule can bepresent, if desired.

Presently preferred are linking compounds in which each of suchfunctional radicals is an amino radical. Both acyclic and cyclic diaminocompounds can be used.

Examples of suitable aliphatic primary diamines include alkylene primaryamines wherein the alkylene group is propylene, butylene, pentylene,hexylene, nonylene, and the like.

Examples of suitable aliphatic secondary diamines includeCH₃NH(CH₂)₂NH₂, CH₃NH(CH₂)₂NHCH₃, and the like.

Diamino compounds incorporating hydroxylated hydrocarbons can be used.Examples of acyclic such compounds include 1,3-diamino-2-hydroxypropane;1,4-diamino-2,3-dihydroxybutane; 1,5-diamino-2,3,4-trihydroxypentane;1,6-diamino-1,6-dideoxy-D-mannitol (or D-glucitol or D-galactitol),1,6-diamino-2,3,4,5-tetrahydroxy hexane, and the like.

Examples of suitable polyhydroxylated cyclic dimensions include cis ortrans cyclic diamino compounds where the diamines are constrained in aring, such as 1,4-diamino-2,3,5,6-tetrahydroxy cyclohexane, cis andtrans 1,2-diaminocyclohexane, cis and trans 1,2-diaminocyclopentane, andhydroxylated derivatives thereof, such as1,2-diamino-3,4,5,6-tetrahydroxycyclohexane,1,2-diamino-3,4,5-trihydroxy cyclopentane,3,6-diamino-3,6-dideoxy-derivatives of myo-inositol, such as

and the like.

Examples of suitable heterocyclic diamines include piperazine,N,N′-bis(3-aminopropyl) piperazine, derivatives thereof, and the like.

Examples of suitable ether-group containing diamines include3-oxo-1,5-pentanediamine, 3,6-dioxo-1,8-diaminooctane, and the like.

Examples of suitable linking compounds containing both an amino radicaland a carboxyl radical include amino acids, such as sarcosine(N-methylglycine), and alpha amino acids, such as glycine, alanine,glutaric acid, aspartic acid, proline, pipecolinic acid(piperidine-2-carboxylic acid), isopipecolinic acid(piperidine-4-carboxylic acid), glucosaminic acid and derivativesthereof, and the like.

Examples of alpha, omega aminocarboxylic acids (in addition to the aboveidentified amino acids) include 4-aminobutyric acid, 6-aminohexanoicacid, 8-aminooctanoic acid, and the like.

Examples of phosphorous containing difunctional linking compoundsinclude alpha, omega aminoalkyl phosphoric acid, monoesters, such as0-(2-aminoethyl) phosphate disodium salt and the like.

Examples of suitable sulfur containing difunctional linking compoundsinclude alpha, omega aminoalkyl sulfonic acids, such as taurine(2-amino-ethyl sulfonic acid) and the like.

One presently more preferred class of difunctional linking compounds isrepresented by the following generic formula:

wherein:

X is a divalent radical selected from the class consisting of:

 wherein:

R is an alkylene radical containing from 2 through 12 carbon atomsinclusive, or of per-hydroxylated carbocyclic ring, and

R₁ and R₂ are each independently selected from the class consisting ofhydrogen and lower alkyl.

Preferably, in Formula (1), R contains not more than 7 carbon atoms,

X is

 and R₁ and R₂ are each hydrogen, and R contains less than 7 carbonatoms.

Mixtures of different linking compounds can be used, such as linkingcompounds containing a mixture of mono and/or diamines, but suchmixtures are not preferred because of associated problems intransamination control and usage.

Diamines which are characterized by having a large proportion thereofthat exists as a free unprotonataed species at pH values of about 7appear to enhance the present transamination reaction. Ethylene diamine(pK of about 7.6) is presently most preferred for use as the reactivedifunctional amine because of this property.

When, for example, such a linking compound is bonded to a DNA sequenceusing a transamination reaction, as hereinbelow described, thetransamination reaction is carried out so that an amino radical in thelinking compound bonds to the sequence or segment. Then, in theresulting linking group, one functional group remains free to undergofurther reaction. Thus, when the second functional radical is an aminoradical, such radical remains free thereafter to undergo furtherreaction with the fluorescent compound, as hereinbelow described. Whenthe second functional radical is a carboxyl radical, such radicalremains free thereafter to undergo such a further reaction with thefluorescent compound, as hereinbelow described.

(4) The Starting Fluorescent Compound

The starting fluorescent compounds employed in the practice of thisinvention each incorporate at least one fluorophore substituent (orgroup) per molecule and also one functional (i.e., reactive) substituent(or group) per molecule.

The functional substituent is chosen so as to be reactive with thesecond functional substituent remaining incorporated into a linkinggroup in a transaminated polynucleotide (such as is prepared asdescribed herein.) The linking group is derived from a linking compound(as above described).

For example, in a starting fluorescent compound, the reactivesubstituent can be chosen to be reactive with an amino substituent (asabove defined), or with a carboxyl substituent which is in the acid orthe salt form (as above defined).

For purposes of reactivity with such an amino substituent in a linkinggroup (using a reaction as hereinbelow described), the reactivesubstituent of the fluorescent compound can be a convenientamine-reactive functionality, such as a carboxyl substituent that is inthe acid or salt form (such as above defined), an aldehyde radical orthe like. A presently preferred such reactive substituent is selectedfrom, and exemplified by, the group consisting of isothiocyanates,N-hydroxysuccinimide esters, sulfonyl chlorides, carboxylic acid azidesand the like.

For purposes of reactivity with such a carboxyl substituent in a linkinggroup, the reactive substituent of the fluorescent compound can be aconvenient carboxyl-reactive functionality, such as an amino substituentwhich is in a primary or a secondary form (such as above defined) or thelike. A presently preferred such reactive substituent is a primary aminosubstituent.

The reactive substituent can also sometimes be, for example, a thiol, aphosphate ester, or the like, the choice, depending upon the nature ofthe reaction substituent that is present in a linking group.

In general, any fluorophore substituent or group can be employed in astarting fluorescent compound. If more than one fluorophore substituentper fluorescent compound is used, then it is presently preferred thateach fluorophore substituent be similar or identical in structure toothers thereof in a single fluorescent compound. A present preference isto employ fluorescent compounds containing about 1 to about 3fluorophore substituents per fluorescent compound molecule and mostpreferably a fluorescent compound contains only one fluorophoresubstituent per fluorescent compound molecule.

Preferably, a starting fluorescent compound has a molecular weight whichis not more than about 5000 and more preferably not more than about 1000because larger molecular weights may possibly have an adverse effectupon the hybridization capacity of a product probe with a complementarytarget sequence.

For reasons of detectability, it is presently preferred that a startingfluorescent compound and the fluorophore groups therein have anextinction coefficient of at least about 6,000 M⁻¹ cm⁻¹ (and preferablyat least about 10,000 M⁻¹ cm⁻¹) in the wavelength region of theexcitation light incident on a given specimen, and also a quantum yieldof at least about 0.02. The term “extinction coefficient” is used hereinin its conventional sense to mean the absorbance of a 1 molar (M)solution of the fluorescent compound contained in a 1 centimeter (cm)path length cuvette. Similarly, the term “quantum yield” is used hereinin its conventional sense to mean the number of photons emitted by afluorophore per the number of photons absorbed by that fluorophore.

Exemplary and presently preferred starting fluorescent compounds areshown in Table II below.

TABLE II EXEMPLARY STARTING FLUORESCENT COMPOUNDS¹ 1.7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA) 2.sulforhodamine 101 sulfonyl chloride; also known by the name Texas Red ™or Texas Red ™ sulfonyl chloride (Tx Rd) 3. 5-(and-6)-carboxyrhodamine101, succinimidyl ester; also known by the name5-(and-6)-carboxy-X-rhodamine, succinimidyl ester (CXR)² 4. Lissaminerhodamine B sulfonyl Chloride (LisR) 5. 5-(and-6)-carboxyfluorescein,succinimidyl ester (CFI)² 6. fluorescein-5-isothiocyanate (FITC)² 7.7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester (DECCA) 8.tetramethylrhodamine-5-(and-6)-isothiocyanate (TRlTC)² 9.5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (CTMR)² 10.7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (HCCA) 11.6-[fluorescein-5-(and-6)-carboxamido]hexanoic acid (FCHA)² 12.N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3- indacenepropionicacid, succinimidyl ester; also known by the name5,7-dimethylBODIPY ®propionic acid, succinimidyl ester (DMBP) 13.“activated fluorescein derivative” (FAP); this compound consists of afluorescein nucleus connected through a spacer group to an N-hydroxysuccinimide ester reactive group and desgnated as “FAP” by themanufacturer, Molecular Probes, Inc. 14. eosin-5-isothiooyanate (EITC)²15. erythrosin-5-isothiocyanate (ErITC)² 16. Cascade ™ Blue acetylazide(CBAA); which is the O-acetylazide derivative of1-hydroxy-3,6,8-pyrenetrisulfonic acid ¹Abbreviations used to refer tothese compounds are enclosed within parentheses. For some coumpoundsalternative names, including trademarked names, are provided.  ™ refersto trademarks of Molecular Probes, Inc. The term “succinimidyl ester”refers to the ester formed between a carboxylic acid substituent of thefluorophore and N-hydroxysuccinimide, and is also referred to as an“N-hydroxysuccinimide # ester” or an “N-hydroxysuccinimidyl ester” or an“NHS ester”. ²Certain fluorescein and rhodamine derivatives containreactive substituents (carboxy or isothiocyanate) attached to either the5- or 6-positions. These compounds can be obtained as mixtures of thetwo isomers, designated as “5-(and-6)”, or in some cases as the purifiedisomers. The labeling and fluorescent properties are not expected tovary greatly between isomers or between a specific isomer and themixture. The isomers or mixtures designated above were those used in #labeling experiments (see Examples).

All fluorescent compounds in Table II were obtained from MolecularProbes, Inc. Eugene, Oreg.

As those skilled in the art will readily appreciate, a startingfluorescent compound is preferably selected for use in making a givenproduct probe composition which will produce, under conditions offluorophore excitement in a single specimen, emitted light of a colorwhich contrasts with the color of the light emitted by the fluorophoregroup-containing label portion of any concurrently or sequentially usedother probe or probe composition which is targeted to the same orrelated karyotype, such as a genome, a specific chromosome, or aspecific region of a chromosome, or the like that is within the genomeinvolved.

(C) Probe Production

(1) General

Primarily because of their characteristically relatively large typicalsize and also their random size characteristics, the individual startingpolynucleotides. (which typically have average sizes the range of about50 to about 4000 bp) of a the starting specific chromosome, orchromosomal regions tend to display relatively poor hybrid formingcapacity (after being labeled).

Also, previously known prior art chemical synthetic methods for joininglabel moieties to a nucleotide sequence, particularly afluorophore-containing label moiety, tend to result in problems ofcontrolling the location and the number of label moieties per sequence,and also in problems of sequence alteration. These problems canadversely affect the resulting probe hybridization capacity with thedesired selected chromosomal target materials, and also, ultimately, thedetection by staining of specific chromosomal DNA present in a specimen.

It has now been discovered that these problems can be overcome, and thata direct label probe composition of excellent hybrid forming capacityand probe performance characteristics exists for preselected individualchromosome or chromosome region selective staining purposes in in situhybridization, when a probe composition is comprised of a mixture of DNAsegments such as is described herein wherein the segments are specificto a preselected chromosome or a preselected chromosome region, andwherein the segments are chemically bound to fluorophore groups throughlinking groups.

Various procedures can be employed to prepare such a probe composition.A presently preferred and illustrative preparation procedure is nowdescribed in which the following procedural steps are carried out:

(a) Fragmenting (i.e., disrupting) DNA sequences that are specific toone preselected chromosome or preselected chromosome region into DNAfragments (or segments);

(b) Transaminating deoxycytidine nucleotides existing in the sequences(and consequently also in the derived segments) with a linking compound(as above described); and

(c) Covalently linking (i.e., bonding) residual radicals of the soproduced transaminated linking groups with a fluorescent compound (asabove described).

While step (b), or steps (b) and (c), can precede step (a), it ispresently preferred for step (a) to precede step (b). In the followingdescription, the foregoing (a), (b), (c) step sequence order is used forpresent organization purposes.

Other combinations and variations of such step sequences are alsofeasible for use in preparing a probe composition of this invention. Forexample, one can covalently bond the fluorescent compound to the linkingcompound, then transaminate and finally fragment the DNA sequences(using step conditions similar to those herein provided); however, thisprocedure tends to result in low yields due to the lower solubility ofthe fluorophore group in the transamination reaction.

(2) Fragmenting

The DNA segments are derived from a particular preselected startingchromosomal DNA or starting regional chromosomal DNA (as abovecharacterized) by fragmenting. Before fragmenting, the starting DNApreferably has an average size polynucleotide of at least about 150 bp.After fragmenting, the DNA segments preferably have an average size thatis within a range of about 150 to about 600 bp with a presently morepreferred average size being about 200 to about 400 bp, and a presentlymost preferred average segment size being about 300 bp. Each of thesesegment fragments is believed to be complementary to a segmental portionexisting in one or more DNA sequences which occur in the particularpreselected chromosome or preselected chromosome region.

The number of fragments derived from fragmenting such starting specificchromosomal or regional chromosomal DNA sequences in any given instanceis unknown, probably variable from one batch to another, and large.Also, the structure of the nucleotide sequences of the individualfragments is unknown and probably variable from one batch to another. Infact, a mixture of such segments from DNA sequences of a singlechromosome contains thousands, if not tens of thousands, of differentlystructured segments. For reasons associated with the capacity of aproduct probe composition to stain DNA of an individual preselectedchromosome or chromosome region, and the brightness of the fluorescencein hybrids formed therewith, it is presently believed to be desirable toutilize DNA fragments having a relatively uniform average size in theranges above indicated.

As those skilled in the art will readily appreciate, these DNA fragmentscan be formed from starting specific chromosomal DNA sequences byvarious known techniques, including, for example, enzyme treatment, aswith restriction enzymes, or polymerases, limited DNase I digestion,limited mung bean nuclease digestion, sonication, shearing of DNA in aFrench press, shearing of DNA through a narrow-gauge needle, and thelike.

However, it is presently greatly preferred to form such DNA fragments bysonication of a starting specific chromosomal DNA. Sonication can becarried out by any convenient procedure. Presently preferred sonicationconditions utilize an aqueous dispersion of starting specificchromosomal DNA that is preferably in the range of about 0.05 to about 4mg per ml, although smaller and larger such concentrations can beemployed. The ultrasonic frequency applied is preferably in the range ofabout 20,000 cycles per second and is applied for a total time in therange of about 1 to about 10 minutes with the tube containing the samplepreferably immersed in a cooled bath to reduce heating of the sample.Suitable cooled baths include ice baths and baths containing dry ice andethanol. Energy density applied to the DNA sequence material undergoingsuch ultrasonic processing is variable. For example, in the case of aBranson Sonifier Model 450 (Danbury, Conn.) with the microtip locatedabout 2 to about 5 mm from the bottom of the tube in an aqueoussolution, a suitable output power is in the range of about 25 to about30 watts. Preferably, such ultrasonic energy is applied using about an80% on time, and, correspondingly, about a 20% off time, for a totaltime of about 5 minutes, such as below exemplified herein.

Preferably, the starting specific chromosomal DNA is fragmented beforethe component sequences are subjected to transamination.

Regardless of the method of fragmenting, the result of fragmenting theplurality of sequences comprising specific chromosomal DNA is to producea profusion of fragments of such starting DNA sequences. This profusioncomprises DNA segments that individually occur at each one of aplurality of different locations in an individual preselected chromosomeor chromosome region. The fragmented DNA segments are thus mixtures thatare derived from the sequential starting DNA and that are approximatelyrepresentative thereof.

Obviously, if the starting chromosomally or regionally specific DNA isobtained, for example, from a commercial source in an already suitablyfragmented state, then a separate fragmenting step is not needed beforea subsequent transaminating processing step is undertaken.

(3) Transamination

In the transamination, a minor fraction of the total deoxycytidine basesthat are contained in the starting specific chromosomal DNA sequencesand segments thereof become transaminated with an amino group of adifunctional linking compound (as above described) in the carbon 4 (C-4)atom position of the amino group of cytosine sites (i.e., deoxycytidinenucleotides). The extent of such transamination is such that betweenabout 1 and about 30 mole percent of all deoxycytidine nucleotides thatare present in a starting mixture of DNA segments that is representativeof total genomic DNA of a given genome are thus substituted by such alinking group, and preferably about 2 to about 24 mole percent thereof.Expressed alternatively, about 0.2 to about 8 mole percent of allnucleotides contained in such a mixture of starting DNA sequences or DNAfragments are thus transaminated and preferably about 0.5 to about 6mole percent. All such transaminations involve substantially onlydeoxycytidine nucleotides.

The most effective percentage of amination in any given instance istypically influenced by the particular fluorescent label moiety used.Since the average number of base pairs present in a sequence ispreferably at least about 150, as above indicated, each sequence is thuspreferably substituted by at least about one such linking group duringthe transamination procedure, as desired. Transamination to a greaterextent seems to increase the potential for adversely affecting thespecificity of product probes (subsequently) labeled with somefluorophores, such as FITC and TXRd, for example. High amination levelsdo not affect as greatly the specificity of CTMR and DECCA labeledprobes, for example. Transamination to a lesser extent seems toadversely affect the brightness of fluorescent light generated indetecting product hybrids.

The transamination is conveniently accomplished under aqueous liquidphase conditions in the presence of a bisulfite catalyst. Theconcentration of DNA sequences (or segment fragments) is conveniently inthe range of about 0.1 to about 1 mg per ml, the concentration ofbisulfite anions is conveniently in the range of about 0.4 to about 1.4moles per liter, the concentration of linking compound is convenientlyin the range of about 1 to about 5 moles per liter, the pH isconveniently in the range of about 4.5 to about 8.0, the temperature isconveniently in the range of about 20 to about 45° C., and thecontacting time is typically and exemplarily in the range of about 3 toabout 72 hours (depending upon the amination level desired).

In the present transamination procedure, the bisulfite is convenientlyintroduced in the form of an alkali metal salt, with sodium andpotassium being preferred alkali metals.

At the time of transamination, the linking compound is dissolved in theaqueous transaminating medium.

Preferably before, and also during the transamination, the DNA sequencesor segments are preferably denatured, for example, by a preliminaryboiling of DNA sequences or segments in water, such as for a time ofabout 1 to about 10 minutes followed by chilling to a temperature belowabout 4° C. (presently preferred), or by carrying out the transaminationin the presence of a chaotrope, or by a combination of both procedures.

7. Sequences obtained from the preparation procedure described incopending application Bittner et al. U.S. Ser. No. 07/762,912, publishedas PCT Patent Application WO 93/0246, on Apr. 1, 1993, filed on evendate herewith. The teachings of this application are incorporated hereinby reference. Such sequences are presently preferred for use in thepresent invention.

The complexity of a DNA fragment mixture is illustrated and exemplifiedby the extent of amination achieved after a preliminary denaturing ofthe mixture DNA fragments by boiling. Since transamination only occursat an appreciable rate on single stranded DNA, and since the reformationrate is slow in proportion to the complexity of the DNA, the specificchromosomal DNA is found to aminate to a lesser extent than ischaracteristic for a relatively more complex DNA mixture.

During the transamination with the bisulfite catalyst, reactionvariables as above identified can be varied within the ranges indicatedto achieve a desired degree of transamination with a given linkingcompound reactant.

The present transamination reaction is carried out or continued until adesired extent of transamination of the starting DNA sequence or segmentmixture is obtained. In general, the maximum extent of transamination isdetermined by the level of transamination which causes, or begins tocause, either an adverse effect upon the complementary character of thenucleotide sequence or segments involved, or an increase in the amountof non-specific association of the subsequently labeled probe withnon-target DNA or other cellular components, such as exist in aspecimen, slide preparation or the like, during an in situ hybridizationusing a probe composition of this invention. There is evidentlytypically and preferably present in a transaminated product only a lowmole percentage of totally unlabeled DNA sequences. The minimum level oftransamination achieved in any given instance is determined by theobjective of achieving substantially complete staining of a preselectedchromosome or region of a chromosome should such be present in a givenspecimen. Even low amination levels do provide such result.

Low levels of amination may be desired so that after staining, apreselected chromosome or region of a chromosome can be observed withoutobscuring another stain (and associated specimen bodies) present in agiven specimen.

The intensity of a specific chromosomal stain achievable in a specimenwith a specific chromosomal staining probe composition of this inventioncan be readily regulated or reduced, if desired, so as to achieve adesired stain coloration intensity in a given specimen or the like. Sucha reduction can be achieved by various techniques, such as, for example,(a) by lowering the extent of linking group transamination, therebyultimately reducing the quantity of labeled nucleotide per unit weightthat are present in a staining probe composition of this invention, (b)by diluting a transaminated mixture with a starting unlabeled DNAsegment mixture, thereby introducing a diluent DNA into the probecomposition, (c) by reducing the amount of probe present in a givenhybridization solution, or the like. The fluorescent intensity of alabeled probe composition can be reduced by the addition of a preferablyfragmented (as above described herein) unlabeled starting total genomicDNA thereto, such genomic DNA being of the genome from which thepreselected chromosome was taken, prior to the time when fluorescentcompound bonding is performed (as below further described).

The minimum level of transamination practiced in any given instance isconveniently determined by the desire to transaminate at least apredetermined mole percentage of the total deoxycytidine nucleotidespresent in the starting DNA, such as a fragmented DNA segment mixture.

A mixture resulting from a transamination procedure that is in accordwith the teachings of the present invention can be conventionallyfurther processed. A present preference is to dialyze such a productmixture, against a dilute aqueous buffer, such as sodium borate,tris(hydroxymethyl)aminomethane (TRIS), or the like at a pH of about 8using a conventional dialyzing membrane and ambient temperatures.

The resulting mixture of transaminated nucleotide sequences or segmentsis then conveniently precipitated from the so dialyzed mixture, and thesequence is then separated from the supernatant by filtration,centrifugation, or the like.

Enzymatic techniques for obtaining aminated DNA are described inAnalytical Biochemistry 157:199-207 (1986). The transaminated DNAsequences are covalently linked to any of a number of fluorescentcompounds that have a reactive functional group capable of covalent bondformation with the transaminated DNA sequence.

(4) Fluorescent Compound Bonding

A resulting transaminated and amine substituted nucleotide derivative isthen available for covalently bonding with a reactive fluorophoresubstituent-containing fluorescent compound, such as above described,with such compound reacting with a terminal functional substituentassociated with the residue of the linking compound (i.e., the linkinggroup) that has now been transaminated into a deoxycytidine moiety asabove described. The number of fluorophore substituent-containingfluorescent compounds thus reacted per sequence or segment molecule iseasily controlled. Preferably, starting DNA sequences are fragmentedbefore being bound to fluorophore groups. Consequently, in a productprobe composition, the number of label groups per DNA molecule isregulatable, as desired. The nucleotide sequence of each segment in theresulting product probe composition is believed to be substantiallyidentical to that existing in a starting DNA mixture except for theadded presence of the transaminated linking groups and the covalentlybonded fluorophore groups.

The covalent linking or bonding of fluorescent compound to a terminalradical of a linking group in, for example, transaminated DNA segmentsis conveniently carried out under aqueous liquid phase conditions usinga temperature in the range of about 4 to about 50° C., a concentrationof transaminated DNA in the range of about 10 to about 500 μg per ml, anear-neutral pH for reactions of N-hydroxysuccinimide esters (e.g., pHof about 6 to 8) and an alkaline pH for reactions of isothiocyanates andsulfonic acid chloride (e.g., pH 8.5-9.5) and a time which is typicallyand exemplarily in the range of about 2 to about 18 hours.

Typically and preferably, the quantity of the starting fluorescentcompound present is sufficient to provide a substantial molar excessrelative to the total quantity of linking groups that are estimated tobe present in the transaminated DNA sequences. In any given situation,an optimized molar excess can be conveniently determined relative to theconcentration of the transaminated deoxycytidine nucleotide residuespresent in the fragments.

While theoretically the amount of fluorescent labeling compound onlyneeds to equal the amount of transaminated deoxycytidine in the probeDNA, an excess is usually greatly preferred since some of thefluorescent compound molecules react by other routes which do not leadto attachment of label to DNA, such as reaction with water (hydrolysis),thereby to render a labeling compound nonreactive with aliphatic aminegroups. Excess labeling compound is also used to increase the rate ofreaction with the transaminated deoxycytidine so that the reaction iscomplete in a shorter amount of time. A larger excess is required forlabeling compounds more sensitive to hydrolysis, such asN-hydroxysuccinimide esters and sulfonic acid chlorides, relative tocompounds less sensitive to hydrolysis such as isothiocyanates. While asmall excess of labeling compound to transaminated deoxycytidines maylead to low percentages of probe labeling, a large excess of labelingcompound can be advantageous in providing high labeling percentages.Labeling compound quantities in excess of that required to achievecomplete labeling is not disadvantageous to the labeling reaction. Veryhigh amounts of labeling compound, however, can lead to post-reactionpurification problems since substantially all of the unreactedfluorescent compound should be removed prior to using the product probecomposition in in situ hybridization or the like. Therefore, very largeexcesses of fluorescent compounds are to be avoided, such as excessesgreater than about a 250 fold molar excess.

When, for example, the fluorescent labeling compound is a succinimidylderivative (that is, an N-hydroxy succinimide ester of a carboxylsubstituted fluorophore or the like), or a corresponding sulfonic acidchloride derivative, then the transaminated DNA sequences containing theresidual linking compound are conveniently reacted with about a 100 toabout a 200 fold molar excess of the fluorescent labeling compound,relative to the intermediate transaminated nucleotides. For convenienceherein, it is assumed that about 5% of total nucleotides aretransaminated.

When, for example, the fluorescent labeling compound is anisothiocyanate derivative, then the transaminated DNA sequences areconveniently reacted with about a 50-fold molar excess of thefluorescent labeling compound.

In the reaction which occurs, covalent bonding is believed to occurbetween the reactive group of a starting fluorescent compound and theterminal group of a transaminated linking group (derived from a linkingcompound) that is associated with a DNA sequence. Preferably, at leastone terminal group of one linking group per molecule is reacted with thefluorescent compound employed. Typically, about 10 to about 100 molepercent of the terminal sites (or terminal functional substituents) ofthe linking groups are reacted (covalently bonded) and thusfluorescently labeled. The extent of reaction between linking groups andfluorescent compounds appears to be influenced by the nature of thefluorophore substituent(s) present in a particular fluorescent compound.Preferably, for efficiency reasons, at least about 70 mole percent ofthe linking groups of a given transaminated DNA composition are solabeled, and most preferably at least about 90 mole percent thereof areso labeled. Thus, in general, preferably about 0.3 to about 6 molepercent of the total nucleotides present in such a starting mixture ofDNA segments are fluorescently labeled.

Residual labeling compound not covalently attached to the probes at theend of the reaction time can be removed by a variety of methods, such asprecipitation of the DNA, gel permeation chromatography, affinitychromatography, dialysis, gel electrophoresis, and combinations of thesemethods. The number and types of procedures combined to purify labeledprobes depends upon the size of the labeling compound, including thesize of aggregates of these labeling compounds, and their ability tononcovalently bond with the labeled DNA, such as through ionic andhydrophobic interactions. One procedure which provides adequate removalof unreacted labeling compound involves an ethanol precipitation stepfollowed by a gel permeation chromatography step followed by a secondethanol precipitation step. The resulting precipitated labeled probe canbe dissolved in water to provide a stock solution of probe which may beused directly in in situ hybridization reactions when combined with theother hybridization components (e.g., formamide, dextran sulfate,buffer, and like known specific probe components).

The resulting reaction product of the transaminated DNA sequences andthe selected fluorescent compound comprises a probe composition of thisinvention.

(D) Direct Label Probe Composition

(1) Probe Compositions

Thus, there is provided a class of direct label probe compositionwherein DNA segments are bound to fluorophore groups. Such a probecomposition considered as a unit is suitable for use in specificchromosomal or specific regional chromosome DNA staining and fordetecting by in situ hybridization or the like the presence in aspecimen of a DNA target region that is either a preselected chromosomeor a preselected chromosome region, as the case may be. Such a probecomposition contains a plurality of different DNA segments thatindividually occur throughout a preselected target region (either asingle chromosome or a single chromosome region). These DNA segments aresubstituted on about 1 to about 30 mole percent of the totaldeoxycytidine nucleotides thereof with a linking group structure whichinitially retains a terminal functional (or reactive) group. At leastabout 10 mole percent of all such retained terminal functional groupshave each been covalently bound to an individual fluorophore radicalcontaining group. Individual ones of such so labeled plurality of DNAsegments that thus comprise such a probe composition of this inventionare hybridizable to complementary DNA segmental portions occurring inDNA sequences of the preselected target region.

Preferably, in a probe composition of this invention, this difunctionallinking group is characterized by the formula:

wherein:

X is selected from the divalent group consisting of:

R₁ and R₂ are each independently selected from the group consisting ofhydrogen and lower alkyl;

m and n are each an independently selected integer of 1 through 6inclusive; and

p is the integer 0 or 1, and wherein the group:

 is transaminated to a deoxycytidine nucleotide of one said DNA segmentand the group —X— is covalently bonded to one said label moiety.

Thus, a direct label probe composition of this invention comprises amixture of DNA segments which are derived from, and are approximately arepresentation of, the DNA of a given preselected chromosome or a givenpreselected chromosome region. A presently preferred class ofchromosomes comprise those of the human genome. The DNA segments of sucha mixture are chemically bound through the intervening linking groups tofluorophore groups. The characteristics of probe compositions of thisinvention are summarized for convenience in Table III below:

TABLE III Characteristics of Probe Compositions of the Invention Ranges(approximate) Presently Variable Broad Preferred Most Preferred 1.Mixture of DNA Segments 1.1 Individual 150-600 200-400 about −300segment average size in bp 1.2 Mole percent  1-30  2-24  4-20 of alldeoxy- cytidines in DNA segments substi- tuted by linking group 2.Difunctional linking group 2.1 No. of carbon  2-20 2-6 about 2 atoms pergroup 2.2 No. of linking 0.4-45   1-24  3-15 groups per DNA segment 3.Flourophore group 3.1 No. of fluoro-   1-4.5 about 1 about 1 phoreradicals per flourophore group 3.2 Mole percent  10-100  70-100  90-100of all linking groups sub- stituted by fluorophore group 3.3 No. offluoro- 0.04-450  0.7-24  2.7-15  phore per segment

(2) Formulations of Probe Compositions

The probe compositions of this invention can be utilized, that is, made,sold, and used, in various forms, including dry solid form, aqueoussolutions, and aqueous formulations that are adapted for direct usage ina hybridization procedure.

In aqueous media, a probe composition of the invention is preferably ina substantially completely dissolved form. As those skilled in the artof DNA probes will appreciate, the content of an aqueous formulation ofa probe composition (including a hybridization solution) can vary widelydepending upon many variables and objectives. For an illustrativeexample, one suitable class of hybridization solutions has a compositionas characterized in Table IV below:

TABLE IV Illustrative Class of Probe Composition Suitable For Use In InSitu Hybridization Ranges (approximate) Component Broad Preferred 1.Probe Composition 1-200 ng/μl of the Invention 2-200 ng/μl paints 5-60ng/μl - paints 1-30 ng/μl cens 1-10 ng/μl - cens 2. Denaturant 0-8%(v/v) 50-80% (v/v) 3. Hybridization Rate 0-15% (w/v) 8-12% (w/v)Promoter 4. Buffer Salt(s) 5-100 mM 10-50 mM 5. Hybrid Stabilizer 0.05mM-1M 0.2-0.5 M salt 6. Blocking DNA 0-10 μg/ml 0.1-0.3 μg Cot-1 DNA0.2-0.7 μg human placental DNA 7. Water Balance Balance

In such a probe composition of Table IV, the denaturant functions topromote denaturation of the DNA segments present in a probe composition.Such promoted denaturation is desirable because it lowers thetemperature employed for denaturation and for hybridization. While thevarious denaturants known to the prior art can be employed, a presentlymost preferred denaturant is formamide.

Similarly, the various hybridization rate promoters known to the priorart can be used. A presently most preferred hybridization rate promoteris a dextran sulfate.

Similarly, the various water soluble buffer salts known to the prior artcan be used. A present preference is to maintain the pH of a probecomposition solution at a value that is in the range of about 5.5 toabout 8.5. Buffer salts which are suitable for maintaining such a pHinclude, for example, citric acid, tris (hydroxymethyl) amino methane,phosphoric acid, and the like. A presently preferred buffer saltcomposition can comprise citric acid (or sodium citrate).

Similarly, a hybrid stabilizer salt which promotes stabilization ofhybrids formed during a hybridization procedure is desirable. The hybridstabilizer salts known to the prior art can be used, such as NaCl, KCl,MgCl₂, and the like. However, the presently most preferred such salt issodium chloride.

The water used is preferably preliminarily distilled or deionized.

(3) Blocked Probe Compositions

An optional but preferred component of a probe composition such as shownin Table IV is a blocking DNA composition.

The use of a diluent DNA in the manufacture of a probe composition ofthe invention is above discussed (see the above subsection ontransamination) as one means for regulating fluorescent intensity in ahybridized target. A diluent DNA can also function as a blocking DNAcomposition and as a means for regulating fluorescent intensity andhybridizing specificity. Blocked compositions of this invention arepresently preferred. Presently preferred blocking DNA compositions forblocked probe compositions of this invention which are targeted to humanchromosomes or regions of chromosomes include fragmented human placentalDNA and Cot-1.

Cot-1 DNA is obtainable as catalog #52795A from Life Technologies, Inc.,Gaithersburg, Md. Cot-1 DNA is reportedly prepared by the followingprocedure: Mechanically sheared total human genomic DNA is fragmented toan average sequence size of less than 400 bp. This material isdenatured, and then hybridized for a period sufficient to render largefractions of the highly repeated DNA sequences thereof double-stranded.The mixture of double and single-stranded DNA species are then treated(digested) with nuclease S1, a nuclease which specifically degradessingle stranded DNA to mono- and oligo-nucleotides. Undigesteddouble-stranded Cot-1 DNA is recovered from this mixture. Cot-1 DNA isreportedly in the form of segments having an average size of about 191bp.

Thus, such a useful starting blocking DNA class is derived from a totalgenomic DNA, which has been denatured, partially reannealed orre-hybridized, and treated with enzymes, or otherwise processed, toreduce the amount of naturally occurring non-repeated segments therein,by processes known to the prior art.

The total weight of blocking DNA composition that is admixed with agiven probe composition of the invention is preferably in excess of thetotal weight of the probe composition. A resulting mixture of a probecomposition of this invention and a blocking DNA composition canoptionally be subjected to hybridizing conditions for a period of timebefore subsequently being used for hybridization with a target andselective staining. Alternatively one can, if desired, directly use adenatured blocked probe composition for hybridization with a targetunder hybridizing conditions.

A blocking DNA admixed with a probe composition of this inventionprevents repetitive common DNA segments in large part from hybridizingwith chromosomal DNA of a preselected chromosome or chromosomal regionpresent in a specimen. Blocked probe composition are beneficial becausestaining selectively is improved, for example, when specific chromosomalprobe preparations are being used in combination with other specificprobe compositions.

Thus, repetitive sequences in such a specific blocked probe compositionare prevented form hybridizing to the same repetitive sequence thatoccurs in, for example, non-targeted chromosomes. Such a hybridizationalso occurs when the blocking DNA composition is present at the timewhen a blocked probe composition of this invention is being hybridizedunder hybridizing conditions with a target. The hybridization ofblocking DNA composition DNA segments to target complementary segmentalregions probably also occurs. The effect of such hybridizations is toregulate the intensity of the fluorescence produced by a given probecomposition after hybridization to a target composition. The quantity ofblocking DNA composition present is now believed to be inversely relatedto the hybrid intensity observed after hybridization; however, thereappear to be many variables influencing the relationship.

The compositions of Table IV can be prepared, if desired, frompreliminarily prepared precursor compositions which are admixed togetherat the time of usage in a hybridization procedure. Such precursorcompositions can be referred to as a “kit”.

(E) In Situ Hybridization and Staining

(1) General

Probe compositions of this invention are well suited for use inhybridization procedures as stains for respective preselectedchromosomes or chromosome regions.

The invention thus provides a process for identifying a preselectedchromosome or chromosome region present in a specimen. The processinvolves the three sequential steps of (a) contacting a specimenbelieved to contain such a chromosome or chromosome region (includingfragments thereof) under hybridizing conditions with a probe compositionof the invention which will hybridize with the target DNA of achromosome or chromosome region to produce hybrids between the targetDNA and the probe DNA segments present in the probe composition, (b)separating from the resulting specimen residual portions of the probecomposition, and (c) examining the resulting specimen.

The specimen examining can be variously accomplished, for example, witha fluorescent microscope, a flow cytometer, or the like. Examinationinvolves irradiating the resulting specimen with energy which is atleast sufficient to cause fluorophore groups present in the hybrids tofluoresce while concurrently detecting the resulting fluorescent energyso produced. As those skilled in the art will appreciate, depending uponthe particular objectives and conditions utilized, this process ofexamining or identifying can be practiced without the separating step ifthe level of residual probe composition is so low as not to interferewith the examination.

(2) Slide Staining

Advantageously, any convenient or particular in situ hybridizationprocedure can be used in the practice of this aspect of the presentinvention. An in situ hybridization procedure can involve a particularspecimen which contains all or only a fraction of the preselectedchromosome or chromosome region.

Currently, a present preference is to use a probe composition of thisinvention in in situ hybridization procedures of the type that arecommonly and conveniently carried out on specimens which havepreliminarily been prepared and mounted on a slide, such as a slidecomprised of glass or the like. Conventional slide preparationprocedures can be employed, such as taught, for example by: F. T. Bosmanet al. in Genetica 5: 425-433 (1975); and Gall et al. in Proc. Natl.Acad. Sci. USA 64: 600 (1969); and conventional in situ hybridizationprocedures can be employed, such as taught, for example, by B. Bhatt etal. in Nucleic Acids Research 16: 3951-3961 (1988); A. H. N. Hopman etal. in Experimental Cell Research 169: 357-368 (1987); NcNeil et al. inGenet Anal Techn Appl 8: 41-58 (1991); and Tkachuk, D. C. et al. inGenet Anal Techn Appl 8: 67-74 (1991).

To accomplish DNA staining of a preselected chromosome or chromosomeregion in such a slide mounted and processed specimen using a probecomposition of this invention, the following illustrative procedure canbe carried out.

Preferably, such slide mounted specimen is preliminarily processed todehydrate at least partially and also denature at least partially theDNA that is presumed to be present therein. Conventional denaturing anddehydrating procedures can be employed, such as exemplified in theEmbodiments below provided. Thereafter, a sequential hybridization stepsequence typically is carried out under hybridizing conditions. First,the slide mounted specimen is contacted with the probe composition ofthis invention.

Next, the combination of the specimen and the treating probe compositionthat is in contact therewith are subjected to an incubation period whichis typically and preferably in the range of about 60 to about 1,000minutes, though longer and shorter incubation period times can beemployed, if desired. During the incubation period, the temperature ispreferably maintained in the above indicated range of about 30 to about45° C. During this incubation period, the treating probe compositionundergoes hybridization with the genomic DNA that is present in thespecimen.

Next, the resulting hybrid-containing specimen is subjected to a liquidwashing procedure that is adapted to separate therefrom unreacted,residual treating probe composition. Advantageously, washing proceduressimilar to those known to the prior art of in situ hybridization can beused (for example see Bhatt et al in Nucleic Acids Research 16:3951-3961 (1988)). Usually several different wash baths are employedwhich contain NaCl, buffer salts, such as sodium citrate, and the like,and formamide, at various concentrations. Higher formamideconcentrations and lower NaCl concentrations are used for higherstringency (greater ability to remove residual probe DNA). Alsodetergents can be used, especially in the last bath. Stringency is alsoincreased by raising the temperature of the wash baths above roomtemperature.

After soaking in a last wash bath or the like, the slide is allowed todrain preferably in a near vertical position and to be fully orpartially air-dried prior to subsequent adding of a mounting medium andcovering with a coverslip. The mounting medium (usually a solution)conventionally contains glycerol, buffer salts, and an antifade materialwhich reduces the rate of photo-oxidation of the fluorophore labels, asthose skilled in the art will appreciate. A chemical type counterstaincan be incorporated into the mounting medium.

The slides can be viewed after processing immediately under afluorescence microscope and conventional filters, or they can be storedat room temperature for several days or the like before examination.

The resulting slide mounted specimen can be further hybridized withother probe preparations. For example, the coverslip can be removed, theslide can be soaked (immersed) in one of the wash baths to removesurface deposits of the mounting medium. A preferably denatured secondprobe hybridization solution is then applied (as above described) to theslide (the slide is not denatured a second time), the resulting slide isincubated to facilitate hybridization, and then the slide is washed, asdescribed above.

In the case of examining a specific chromosomally stained specimenproduced from a probe composition of this invention, as those skilled inthe art will appreciate, the particular filter used is preferably onewhich is matched to the spectral response characteristics associatedwith the particular fluorophore that is involved as the label moiety ina given probe composition of this invention. Such a filter either iscommercially available, or is readily made by conventional filter-makingor filter assembly technology. The following Table V presentsillustrative specifications for filters and such filters were used withthe fluorophores included in the Examples herein presented:

TABLE V FLUORESCENCE MICROSCOPE FILTER SET NOMINAL SPECIFICATIONS FilterSet Excitation Dichroic Emission Filter Set Fluorophore Filter†Beamsplitter‡ Filter† Ref. # AMCA/ BP 365 (>50)* 395 LP 420 1 DAPI BP360 (50)** 400 BP 450 (50) 2 CBAA BP 360 (50)** 400 BP 425 (35) 11 HCCABP 400 (20)** 440 BP 456 (28) 12 DECCA BP 435 (20)** 475 BP 535 (45) 3BP 432 (20)** 455 BP 478 (32) 13 FITC BP 470 (40)* 510 LP 520 4 BP 485(20)* 510 BP 540 (50) 5 BP 480 (30)** 505 BP 535 (45) 6 BP 496 (24)**515 BP 532 (28) 14 CFI (same as for FITC) DMPB (same as for FITC) FCHA(same as for FITC) FAP (same as for FITC) EITC BP 518 (26)** 545 BP 558(30) 15 ErITC (same filters as EITC, although not optimal for thisfluorophore - should be shifted to longer excitation and emissionwavelengths relative to EITC) TRITC BP 546 (12)* 580 LP 590 7 BP 540(23)** 570 BP 605 (55) 8 CTMR (same as for TRITC) LisR (same as forTRITC or Tx Rd) Tx Rd BP 560 (40)** 595 BP 635 (60) 9 (same as for TxRd) Propidium BP 540 (23)** 565 BP 615 (30) 10 iodide †Wavelength valuesare listed in units of nanometers. Bandpass filters are marked “BP” withthe center of the filter's transmission band listed first and the fullwidth at half maximum enclosed in parenthesis. Long pass filters aremarked “LP” with the transition region between low and high transmissionindicated. ‡Wavelength values are listed in units of nanometers andindicate the region of the filter's transition between high reflectanceand high transmission. *Filter set obtained from Zeiss. **Filter setobtained from Omega Optical.

Those skilled in the art will appreciate that in in situ hybridizationof a slide mounted specimen, the sequence of (a) contacting and (b)separating (as above indicated) can be advantageously carried out morethan once before the step (c) (examining) as above indicated is carriedout. In each such repeat of steps (a) and (b) (each of which isconveniently carried out as above described herein), a different probecomposition is employed, with a probe composition of this inventionbeing employed on one repeat, and with another (different) probecomposition being employed in each of the other repeats, each such otherprobe composition being targeted to a different predetermined fractionalregion of said genome.

(3) Flow Cytometry

The specific chromosomal staining probe compositions of this inventioncan also be used, for another example of utilization, in a procedureutilizing fluorescence activated flow cytometry. For example, initiallychromosomes can be conventionally isolated, for instance, from mitoticcells of a cell culture; see, for example, Carrano et al. in Proc.Nat'l. Acad. Sci. USA 76: 1382-1384 (1979).

Next, an aqueous dispersion of the so isolated chromosomes has admixedtherewith a crosslinking agent for the protein (i.e., histones andnonhistones) present in the chromosomal chromatin with the DNA.Conveniently, the crosslinking agent reacts with a polar group of one ormore polar group containing amino acids present in such protein, suchas, for example, asparate, aspargine, arginine, glutamate, glutamine,histidine, lysine, serine, tyrosine, and tryptophan. The sulfhydro groupof cysteine can also sometimes crosslink. A suitable crosslinking agentand a suitable in situ hybridization procedure are taught, for example,in Van Engh U.S. Pat. No. 4,770,992 (1988).

A probe composition of the invention is admixed with the crosslinked andpreferably denatured chromosomes.

A resulting mixture is preferably subjected to a separation procedure toisolate unhybridized residual probe composition. However, as thoseskilled in the art will appreciate, if the concentration of residualprobe composition is sufficiently low so as not to interfere, orexcessively interfere, with the particular flow cytometric analysiscontemplated, then such separation procedure can be circumvented.

A suitable separation procedure can involve centrifuging. The resultingchromosome cake is resuspended in an aqueous medium. A convenientresuspended concentration is about 5×10⁶ chromosomes/ml. Conveniently,the suspension water has dissolved therein a buffer salt.

A resulting suspension is subjected to flow cytometric analysis using,for example, a dual beam cytometer, for example, such as described inthe aforecited references.

The results so measured can be used, for example, to identifychromosomes based upon the gross specimen morphology, or to correlatethe presence of a specific chromosome stain with the presence of anychromosome material. The correlation is made to discriminate against thebackground which could be confused with the specific stain.

Another technique combining flow cytometric detection with in situhybridization using a probe composition of this invention usesinterphase nuclei in suspensions in the manner taught, for example byTrask, et al. in Hum Genet (1988) 78:251-259.

(4) Probe Mixtures

It is a feature and advantage of the probe compositions of thisinvention that they can be admixed with other probe compositions and thelike without adversely affecting the chemical structure or thefunctional capacity thereof. Thus, even though mixed probe compositionsincorporate complex DNA segment mixtures under hybridizing conditions,these individual segments only hybridize with complementary target DNAso that the desired specific chromosomal staining is achieved. Forexample, prior to the first procedural step above indicated, a probecomposition of this invention, prepared as above described, can beadmixed, if desired, with another probe composition which, for example,contains labeled segments that are hybridizable to specific targetspresent in predetermined chromosomes, or chromosome regions. Such otherprobe compositions should preferably be suitable for usage in in situhybridization under comparable conditions of temperature, time, and thelike (relative to a probe composition of this invention).

For example, such other probe composition can be a direct labeled or anindirect labeled probe composition such as (a) another direct labeledprobe composition of this invention; (b) a probe composition labeled bynick translation of that probe composition's DNA withfluorophore-labeled nucleoside triphosphates as described in Wiegant etal. in Nucleic Acids Research 19: 3237-3241 (1991); (c) an indirectlabeled probe composition prepared by incorporating biotin- ordigoxygenin-containing deoxynucleotide triphosphates into probe DNA asdescribed in a number of publications including Wiegant et al. (op.cited) and Bhatt et al. in Nucleic Acids Research 16: 3951-3961 (1988);and/or (d) the indirect labeled probe compositions which containchemical groups that react in a post hybridization reaction withchemical groups on modified fluorophores to form a bond betweenhybridized probe and fluorophore label, as described by Hopman et al. inExperimental Cell Research 169: 357-368 (1987); and the like.

Such a resulting mixed probe composition can then be used in an in situhybridization procedure in a slide mounted specimen. In this procedure,each chromosomal target present in a specimen is stained with itspreselected probe composition, to the extent, that such target ispresent in the specimen. Residual probes are subsequently separated fromthe resulting specimen in the same washing procedure.

Another feature and advantage of the probe compositions of thisinvention is that they can also be used for successively accomplishedspecific staining of preselected chromosomes even after the completionof a preceding hybridization procedure, such as an in situ hybridizationprocedure wherein a particular different target is involved, or thelike.

Probe compositions of this invention are generally compatible with otherin situ hybridization reagents. However, in the case of two or moreindirect labeled probes in combination, care must be taken that nocomponents are present which will cross-react with each other when inadmixture.

EMBODIMENTS

The present invention is further illustrated by reference to thefollowing examples.

Microscopy was performed on either a Zeiss Axioskop, Axioplan, orAxiophot fluorescence microscope. Filter sets used for viewing specimensstained by in situ hybridization were obtained from either Carl Zeiss,Inc. (Thornwood, N.Y.) or Omega Optical, Inc. (Brattleboro, Vt.) asshown in Table V.

EXAMPLE 1 Isolation of Human Chromosome-Specific DNA Probes

Human chromosome-specific DNA was obtained as recombinant phagelibraries from Lawrence Livermore National Laboratories, constructed asdescribed in Van Dilla, M. A. et al. (Biotechnology 4: 537-552, 1986).Deposits of these libraries have been made to the Lawrence LivermoreNational Laboratories (LLNL). These libraries were amplified by growthon an E. coli host strain. The amplified phage were purified, their DNAwas extracted, and this DNA was digested with the restriction enzymeHind III. Insert DNA was purified away from the lambda vector DNA andcloned into the Hind III site of the plasmid vector pBS (Strategene, LaJolla, Calif.). The resulting plasmids were transformed into an E. colistrain, DH5α (Bethesda Research Libraries, Gaithersburg, Md.), therebycreating recombinant plasmid libraries containing human chromosomesspecific insert DNA.

The phage libraries exemplified herein correspond to ATCC #57754(Chromosome 1); ATCC #57745 (Chromosome 4); ATCC #57701 (Chromosome 6);ATCC #57702 (Chromosome 8) and ATCC #'s 57722 and 57755 (Chromosome 7).The libraries are stored as 1 ml aliquots of frozen cells. These vialshave been used as the primary source for chromosomal DNA. Plasmidlibraries from these phage libraries are used for fermentation.

Bacteria were grown by fermentation. The seed stock obtained fromLawrence Livermore National Laboratories was cultured at 37° C. for 24hr. on 1.6% agar plates containing ampicillin (200 microgram/ml) and YTbroth, which contains 8 grams per liter (g/l) of Bacco Tryptone (Difco),5 g/l of Bacco Yeast Extract (Difco), 15 g/l of Bacto Agar (Difco), and5 g/l of sodium chloride. The cultured cells were harvested with 4 ml ofYT broth containing 16 g/l of Bacto Tryptone (Difco), 10 g/l of BactoYeast Extract (Difco) and 5 g/l of sodium chloride, and 4 ml of 20%glycerol was added to each harvest. The E. coli cell culture was quicklyfrozen in 0.5 ml aliquots by submerging the vials in liquid nitrogen andstored at −80° C. until use.

The fermenter inoculum was prepared in 350 ml by culturing the seedculture in a Casamino Acid medium which contains 13.2 g/l Na₂HPO4-7H₂O,3.0 g/l KH₂PO₄, 0.05 g/l NaCl, 1.0 g/l NH₄Cl, 10.0 g/l Casamino Acids(Difco); 0.03 g/l MgSO₄, 0.004 g/l CaCl₂-2H₂O, 3.0 g/l glucose, 0.025g/l Thiamine-HCl, 0.0054 g/l FeCl₃, 0.0004 g/l ZnSO₄, 0.0007 g/l CoCl₂,0.0007 g/l Na₂MoO₄, 0.0008 g/l CuSO₄, 0.0002 g/l H₂BO₃, and 0.0005 g/lMnSO₄ in a 2 liter baffled shaker flask at pH 7 and 37° C. The 350 mlculture was used to inoculate 4.2 liter of fermentation media containing1% glucose, 13.2 g/l Na₂HPO4-7H₂O, 3.0 g/l KH₂PO₄, 0.05 g/l NaCl, 1.0g/l NH₄Cl, 10.0 g/l Casamino Acids (Difco), 0.03 g/l MgSO₄, 0.004 g/lCaCl₂-2H₂O, 0.025 g/l Thiamine-HCl, 0.0054 g/l FeCl₃, 0.0004 g/l ZnSO₄,0.0007 g/l CoCl₂, 0.0007 g/l Na₂MoO₄, 0.0008 g/l CuSO₄, 0.0002 g/lH₂BO₃, and 0.0005 g/l MnSO₄.

Bacterial cells were harvested employing a membrane cell-concentratorand a high speed centrifuge immediately after completion of thefermentation. The fermented cell broth was concentrated from 5 liters toapproximately 800 ml employing a 0.45 micron (μm) membrane filter (2square feet). The cell concentrate was then centrifuged at 7,000×g for10 minutes in a refrigerated centrifuge. The bacterial cell pellets wererecovered after discarding the supernatant.

The amounts of all of the reagents used in the isolation of the DNA arecalculated relative to the initial wet cell mass of the cell pellet (ingrams). To determine the amounts required, a multiplication factor “M”is calculated, and then each reagent is added at M times a fixed amountof that component. The factor M for a given cell mass is determined bydividing the cell mass, in grams, by 13. Thus, to process 130 grams ofcell mass, an M factor of 10 is applied to the fixed amount of eachindividual reagent.

Plasmid DNA was extracted from bacterial cell pellets. Cell pellets wereresuspended in 40×M milliliters of a solution of 50 mM glucose (filtersterilized), 10 mM NaEDTA (pH 7.5-8.0), and 25 mM Tris-HCl (pH 8.0). Thecells were lysed with vigorous swirling after the addition of 80×Mmilliliters of a solution of 0.2 M NaOH and 1% (w/v) SDS. After a fewminutes the turbidity of this solution decreased, indicating lysis ofthe cells. To the cleared solution was added 60×M milliliters of asolution containing 55.5 ml of glacial acetic acid and 147.5 grams ofpotassium acetate per 500 ml. These solutions were mixed thoroughly,resulting in the production of a flocculent precipitate. The supernatantwas removed from the flocculent precipitate and this supernatant wascentrifuged for 15 minutes at 7000×g to remove residual precipitate.

Nucleic acid was precipitated from the supernatant with one volume ofethanol followed by centrifugation for 10 minutes at 7000×g, and thenucleic acid pellets were resuspended in a total of 7×M milliliters of asolution containing 50 mM Tris-HCl (pH 8.0), 100 mM sodium acetate. Thenucleic acid was then extracted with 1/2 volume of neutralized phenoland 1/2 volume of chloroform and precipitated with two volumes ofethanol. The nucleic acid was resuspended in 4×M milliliters of asolution containing 50 mM Tris-HCl (pH 8.0), 100 mM sodium acetate. A10×M microliter portion of 10 mg/ml pancreatic Ribonuclease A solution(heat treated to inactivate DNase) was added to the resuspended nucleicacid solution. This mixture was allowed to digest for 30 minutes at roomtemperature or overnight at 4° C. An 8×M microliter portion of 20 mg/mlProteinase K solution was then added and incubated at 55° C. for threehours. The DNA solution was then extracted with 1/2 volume ofneutralized phenol and 1/2 volume of chloroform and precipitated withtwo volumes of ethanol. Precipitated DNA was collected by centrifugationfor 15 minutes at 4500×g.

DNA was resuspended in 5.36×M milliliters of water, and then 0.64×Mmilliliters of 5 M NaCl and 2×M milliliters of 50% (w/v)polyethyleneglycol (PEG) (molecular weight 6000-8000) were added,incubated on ice water for one hour and precipitated by centrifugationfor 15 minutes at 4500×g. The DNA was resuspended in 0.5×M millilitersof water and 1/10 volume of 3M sodium acetate and extracted with 1/2volume of neutralized phenol and 1/2 volume of chloroform andprecipitated with two volumes of ethanol. Precipitated DNA was collectedby centrifugation for 15 minutes at 4500×g and dried under vacuum. Thepurified DNA was resuspended in 0.6×M milliliters of deionized water.The DNA concentration was determined by fluorometry.

Finally, the purified DNA was disrupted into small fragments ofapproximately 300 base pairs by sonication using a Branson Sonifier 450(Danbury, Conn.). This size of fragments has been empirically determinedto be the optimum for DNA probes used for in situ hybridization. Fourmilligrams of the purified plasmid DNA prepared above was resuspended in2 ml of water and immersed in a dry ice/ethanol bath to prevent boilingduring sonication. The microtip of the sonication device was immersed inthis solution until the tip was 2-5 mm from the bottom of the tube.Sonication was carried out at an output power of 25-30 watts,discontinuously, with an 80% duty cycle(on 80% of time, off 20% oftime), for a period of 5 minutes. Following sonication, the DNA wasprecipitated by the addition of 0.2 ml of 3 M sodium acetate (pH 5.5)and 4 ml of ethanol. The precipitate was recovered by centrifugation for5 minutes at 8,000×g and vacuum dried.

EXAMPLE 2 Bisulfite Catalyzed Transamination of Probe Precursor DNA

Probe precursor DNA was transaminated by the addition of ethylenediamineto the C4 carbon atom of the base cytosine. This reaction is catalyzedby sodium bisulfite. Different probe DNA precursor DNA sets weretransaminated. Thus, approximately 4 to 24% of the availabledeoxycytidine nucleotide sites are aminated for fluorescent labeling.

To prepare the bisulfite buffer, 1.7 ml of concentrated HCl was slowlyadded to 1 ml deionized H₂O on ice. 1 ml fresh ethylenediamine (Sigmacat. #E-4379) was then slowly added on ice. After dissolution of theethylenediamine, the solution was warmed to room temperature and 0.475sodium metabisulfite (Aldrich Cat. #25,555-6) or sodium bisulfite (EMScience Cat. #SX0345-1) was added. Concentrated HCl was then slowlyadded to the bisulfite mixture until the pH reached 7.0, and the volumeof the solution was adjusted to 5.0 ml.

To transaminated probe precursor DNA, 1 milligram of sonicated DNA wasresuspended in 0.3 ml H₂O. The DNA was denatured by boiling at 100° C.for 5 minutes then quickly chilled in an ice water bath. Thetransamination reaction was initiated by the addition of 0.3 ml of thisDNA solution to 2.7 ml of bisulfite buffer, and the reaction wasincubated at either 25 or 37° C. for several hours to several days toachieve the desired degree of amination (see table below). The DNAsolution was desalted by routine dialysis against 5-10 mM sodium borate(pH 8.0). After dialysis, 0.3 ml of 3 M sodium acetate (pH 5.5) wasadded to the dialysate. The aminated DNA was precipitated with 2.5volumes of ethanol and recovered after centrifugation at 8,000×g for 10minutes. The pellets were vacuum dried and rehydrated at a concentrationof 3 mg/ml DNA. This solution was stored at −20° C. until use.

The extent of transamination of dC was determined by enzymatic digestionof the aminated DNA followed by separation of the resulting nucleosideson an FPLC chromatography system (Pharmacia LKB, Piscataway, N.J.). 5-10μg of aminated DNA was diluted with water to 50 μl and the DNA purifiedon a spin column containing Sephadex C-25 (5Prime→3 Prime, Inc. Paoli,Pa.). The DNA was then dried, resuspended in 12.0 μl H₂O, and 12.5 μl of2×DNase 1 buffer (20 mM Tris, 10 mM MgCl₂, pH 7.5) and 0.5 μl ofdeoxyribonuclease 1 (DNase 1) (BRL, 2 mg/474 μl, >10.000 U/mg) was addedto the DNA and the solution incubated in a 37° C. water bath for 1 hr.50 μl of 2×PD1/alk. phos. buffer (100 mM Tris, 200 mM NaCl, 28 mM MgCl₂,2 mM ZnCl₂, pH 9.0), 19 μl of water, 5.0 μl of phosphodiesterase 1 (PD1)(Pharmacia LKB, 1,000 U/ml dissolved in 1×PD1/alk. phos. buffer), and1.0 μl calf intestinal alkaline phosphatase (Promega, 10,000 U/ml) wasthen added and the solution incubated for an additional 2 hr at 37° C.The digested sample was then applied to a MinoRPC column (Pharmacia LKB)and a linear gradient between buffer A (97.5:2.5 ion-pairingbuffer:methanol, ion-pairing buffer—50 mM KH₂PO₄, 0.05% hexanesulfonicacid, pH 7.0) and buffer B (50:50 ion-pairing buffer:methanol) was usedto elute the sample (a 0.8% increase in buffer B/min at a flow rate of0.37 ml/min until 40% buffer B was reached, followed by a 3% increase inbuffer B/min to 100% buffer B at a flow rate of 0.3 ml/min) whilerecording the DNA elution profile by absorbance. Each of the 4 naturaldeoxynucleosides and the transamination product of deoxycytidine elutedseparately and the amount of deoxycytidine transaminated was determinedfrom the relative areas under the deoxycytidine and transaminateddeoxycytidine peaks in the elution profile. Typical results are listedbelow for two preparations of sonicated plasmid DNA containing sequencesfrom human chromosome #4.

TABLE VI Transmission Reaction Conditions DNA Preparation Temp. (° C.)Time % dC Aminated 1 37 2 day 22 2 37 63 hr 19 2 37 38 hr 19 2 37 15 hr13 2 25 63 hr 12 2 25 38 hr 10 2 25 15 hr 4.6

EXAMPLE 3 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore 7-Amino-4-methylcoumarin-3-acetic Acid,Succinimidyl Ester (AMCA)

Transaminated probe precursor DNA (average length 300 bp, 20% ofdeoxycytosine aminated) specific to human chromosome 1 were conjugatedwith 7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA).Sixty micrograms of transaminated DNA were dried and then resuspended in673 microliters of 200 mM 3-[N-morpholino] propane sulfonic acid (MOPS)as a buffer at pH 7.4. Twenty-six and eight tenths microliters of a 50mM solution of AMCA in dimethyl sulfoxide (a 150 fold molar excess) wasadded to the suspension of transaminated DNA. This reaction proceededwith stirring in darkness at room temperature for approximately 18hours. The excess fluorophore was separated from the labeled DNA bypassing the reaction over a Sephadex C-25 column chat was 28 cm highwith an internal diameter of 1 cm. The desired fraction (the column voidvolume) was eluted with water and dried to reduce the total volume. Twoethanol precipitations of the labeled DNA completed the purification toprovide the AMCA probe. An absorbance spectrum showed that 4.8% of thebases were labeled. In subsequent preparations one of the ethanolprecipitation steps was performed first, followed by the columnpurification step, and the second ethanol precipitation step wasperformed last. Both purification procedures provided good results.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome spreads.The target DNA consisted of cultured normal white blood cells that weretreated to arrest the cells in metaphase. These cells were dropped ontoa glass microscope slide from a distance of about 3 feet to break openthe nuclei. Before hybridizing the slide was placed for 2 minutes in adenaturing solution of 70% formamide/0.3 M NaCl/30 mM sodium citrate (pH7) at 70° C. The slide was then dehydrated by passing through 70%, 85%and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on each slide (10 μl) was 50%formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH 7)/0.5μg sonicated salmon sperm DNA (used as carrier). The concentrations ofblocking DNA (Cot1 DNA or sonicated human placental DNA) and of probeadded to the basic mix were varied. Ten microliters of the completehybridization mixture were denatured by heating to 70° C. for 5 minutesand then allowed to hybridize at 37° C. for one hour. The mix wasapplied directly to the slide, covered with a glass coverslip, andallowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. The antifade solution isdescribed in J. Immuno. Methods 43: 349 (1981) and is made as follows:100 milligrams of p-phenylenediamine dihydrochloride is dissolved in 10milliliters of phosphate buffered saline solution. The pH of thissolution is adjusted to pH 8 with a bicarbonate buffer solution preparedby adding 0.42 g NaHCO₃ to 10 milliliters of water then adjusting the pHto 9.0 by the addition of 50% (w/v) NaOH. The pH adjusted solution ofp-phenylenediamine dihydrochloride is added to 90 milliliters ofglycerol and the resulting solution is filtered through a 0.22 μmfiltration device. This solution is stored in the dark at −20° C. Theantifade solution optionally contained 0.2 μg propidium iodide/ml as ageneral chromosome stain to permit the visualization of all chromosomes.The slide was then viewed with a fluorescence microscope using filterset #1 or #2 for the AMCA and set #10 for the propidium iodide.

TABLE VII Qualitative Results: [Blocking DNA]; Visual Description [AMCAProbe] P = placental, slide under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity 100 2.25P − 316 2.25P +++ + 5002.25P +++ ++ 1,000 2.25P +++ ++ 1,000 6.75P +++ +++ 100 1.3C +/− +/− 3161.3C +++ ++ 1,000 1.3C ++ +++ Code: Specificity: (−) none apparent, (+)small amount of specificity, (++) reasonable specificity, (+++) goodspecificity Intensity: (−) not visible, (+) barely visible, (++) fairlyvisible, (+++) bright, (++++) very bright

AMCA-labeled probes show good specificity with either placental orCot1DNA. Higher probe concentrations are preferable for observing goodfluorescence intensity.

It was often found that adding 6.75 μg rather than 2.25 μg of humanplacental DNA resulted in improved specificity and/or intensity ofprobes to whole chromosomes.

EXAMPLE 4 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore Texas Red Sulfonyl Chloride (TxRd)

Two different transaminated probe precursor DNAs (average length 300 bp,one to 20%, the other to 4.6% of deoxycytosines aminated) specific tohuman chromosome 7 were conjugated with Texas Red Sulfonyl chloride(TxRd). Forty micrograms of each such transaminated DNA were dried andthen resuspended in 270 microliters of 50 mM sodium borate, pH 9.3.Thirty microliters of a 30 mM solution of TxRd in N,N-dimethylformamide(a 150-fold molar excess) was added to the suspensions of transaminatedDNA. These reactions proceeded with stirring in darkness at roomtemperature overnight (approximately 18 hours). The excess fluorophorewas separated from the labeled DNAs by precipitating the DNAs fromethanol. The precipitated materials were resuspended in water and eachpassed over a Sephadex G-25 column that was 28 cm high with an internaldiameter of 1 cm. The desired fractions (the column void volumes) wereeluted with water and dried to reduce the total volumes. A secondethanol precipitation of the labeled DNAs completed the purification.Absorbance spectra of the labeled products showed that 3.2% of the totalnucleotides in the 5% (total nucleotides) aminated DNA preparation werelabeled and that 1.0% of the total nucleotides in the 1.2% (totalnucleotides) aminated DNA preparation were labeled. This procedureprovides a TxRd probe.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were treated to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei and expose the chromosomes. Before hybridizing the slidewas placed for 2 minutes in a denaturing solution of 70% formamide/0.3 HNaCl/30 mM sodium citrate (pH 7) at 70° C. The slide was then dehydratedby passing through 70%, 85% and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by hearing to 70° C.for 5 minutes and then allowed to hybridize au 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (ph 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #9 for Tx Rd and set #1 or #2for DAPI.

TABLE VIII Qualitative Results: [Blocking DNA]; Visual Description [TxRdProbe] P = placental, (slide under microscope 40-100%) (ng/10 μl) C =Cortl (μg/10 μl) Specificity Intensity 5% aminated, 3% Tx Rd-labeled(based upon total nucleotides) probe: 32 2.25P + ++ 100 2.25P + ++ 3162.25P − +++ 1,000 2.25P − ++++ 100 6.75P − ++ 316 6.75P ++ +++ 1,0006.75P − ++++ 100 1.3C ++ ++ 316 1.3C ++ +++ 1,000 1.3C ++ ++++ 1.2%aminated, 1% Tx Rd-labeled (based upon total nucleotides) probe: 1001.3C ++++ +++/++++ 316 1.3C ++++ ++++ Code: Specificity: (−) noneapparent, (+) small amount of specificity, (++) reasonable specificity,(+++) good specificity, (++++) very good specificity. Intensity: (−) notvisible, (+) barely visible, (++) fairly visible, (+++) bright, (++++)very bright.

The data show that Tx Rd-labeled probes perform better when labeled to alower degree. Specificity was much improved when the degree of aminationwas reduced from 20% to 4.6% of the deoxycytosines. Even low probeconcentrations (100 ng/10 μl) provided very good results when using the1% labeled probe.

EXAMPLE 5 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore 5-(and-6)-carboxy-X-rhodamine, SuccinimidylEster (CXR)

Two different transaminated probe precursor DNAs (average length 300 bp,one to 20%, the other to 4.6% of deoxycytosines aminated) specific tohuman chromosome 4 were conjugated with 5-(and-6)-carboxy-X-rhodamine,succinimidyl ester (CXR). Thirty-five micrograms of each suchtransaminated DNA were dried and then resuspended in 368 microliters of200 mM MOPS, pH 7.4. Thirty-one and eight tenths microliters of a 25 mMsolution of CXR in N,N-dimethylformamide (a 150-fold molar excess) wasadded to each suspension of transaminated DNA. These reactions proceededwith stirring in darkness at room temperature overnight (approximately18 hours). The excess fluorophore was separated by precipitating thelabeled DNAs from ethanol. The precipitated materials were resuspendedin water and each passed over a Sephadex G-25 column that was 28 cm highwith an internal diameter of 1 cm. The desired fractions (the columnvoid volumes) were eluted with water and dried to reduce the totalvolumes. A second ethanol precipitation of the labeled DNAs completedthe purification. Absorbance spectra of the labeled products showed that4.9% of the total nucleotides in the 5% (total nucleotides) aminated DNApreparation were labeled and that 1.5% of the total nucleotides in the1.2% (total nucleotides) aminated DNA preparation were labeled.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were treated to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from a distance of about 3 feet tobreak open the nuclei. Before hybridizing the slide was placed for 2minutes in a denaturing solution of 70% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85% and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by hearing to 70° C.for 5 minutes and then allowed to hybridize at 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the race of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #9 for CXR and set #1 or #2 forDAPI.

TABLE IX Qualitative results: [Blocking DNA]; Visual Description[CXR-Probe] P = placental, (slide under microscope 40-100%) (ng/10 μl) C= Cotl (μg/10 μl) Specificity Intensity 5% aminated, 4.9% CXR-labeled(based upon total nucleotides) probe: 100 1.3C − + 316 1.3C +++ +++1,000 1.3C ++ +++ 1.2% aminated, 1.5% CXR-labeled (based upon totalnucleotides) probe: 100 1.3C ++++ +++ 316 1.3C ++++ +++ 1,000 1.3C +++++++ Code: Specificity: (−) none apparent, (+) small amount ofspecificity, (++) reasonable specificity, (+++) good specificity, (++++)very good specificity. Intensity: (−) not visible, (+) barely visible,(++) fairly visible, (+++) bright, (++++) very bright.

The data show that CXR-labeled probes perform better when labeled to alower degree. Specificity was improved when the degree of amination wasreduced from 20% to 4.6% of the deoxycytosines. Even low probeconcentrations (100 ng/10 μl) provided good results when using the 1.5%(total nucleotides) labeled probe.

EXAMPLE 6 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore Lissamine Rhodamine B Sulfonyl Chloride (LisR)

Transaminated probe precursor DNA (average length 300 bp, 20% ofdeoxycytosines aminated) specific to human chromosome 4 were conjugatedwith lissamine rhodamine B sulfonyl chloride (LisR). Thirty microgramsof transaminated DNA were dried and then resuspended in 373 microlitersof 50 mM sodium borate, pH 9.3. Twenty-seven and three tenthsmicroliters of a 25 mM solution of LisR in N,N-dimethylformamide (a150-fold molar excess) was added to the suspension of transaminated DNA.This reaction proceeded with stirring in darkness at room temperatureovernight (approximately 18 hours). The excess fluorophore was separatedfrom the labeled DNA first by an ethanol precipitation. The precipitatedmaterial was resuspended in water and passed over a Sephadex C-25 columnthat was 28 cm high with an internal diameter of 1 cm. The desiredfraction (the column void volume) was eluted with water and dried toreduce the total volume. A second ethanol precipitation of the labeledDNA completed the purification. An absorbance spectrum showed that 3.8%of the bases were labeled. This procedure provides the LisR probe.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were created to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about 3 feet to break openthe nuclei. Before hybridizing the slide was placed for 2 minutes in adenaturing solution of 70% formamide/0.3 M NaCl/30 mM sodium citrate (pH7) at 70° C. The slide was then dehydrated by passing through 70%, 85%and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by heating to 70° C.for 5 minutes and then allowed to hybridize at 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol1, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #7, #8, or #9 for LisR and set#1 or #2 for DAPI.

TABLE X Qualitative Results: [Blocking DNA]; Visual Description [LisRProbe] P = placental, (slide under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity 100 2.25P − + 316 2.25P + ++ 1,0002.25P + ++ 100 6.75P − + 316 6.75P + ++ 1,000 6.75P + ++ 100 1.3C + ++316 1.3C − +++ 1,000 1.3C − ++++ Code: Specificity: (−) none apparent,(+) small amount of specificity, (++) reasonable specificity, (+++) goodspecificity Intensity: (−) not visible, (+) barely visible, (++) fairlyvisible, (+++) bright, (++++) very bright.

LisR-labeled probes provide some specificity and good intensityrelatively high degree of 5% labeling (total nucleotides).

EXAMPLE 7 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore 5-(and-6)-Carboxytetramethylrhodamine,Succinimidyl Ester (CTMR)

Two different transaminated probe precursor DNAs (average length 300 bp,one to 20%, the other to 4.6% of deoxycytosines aminated) specific tohuman chromosome 4 were conjugated with5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (CTMR). Fiftymicrograms of each such transaminated DNA were dried and thenresuspended in 377 microliters of 200 mM MOPS, pH 7.4. Twenty-two andeight tenths microliters of a 50 mM solution of CTMR inN,N-dimethylformamide (a 150-fold molar excess) was added to eachtransaminated DNA. These reactions proceeded with stirring in darknessat room temperature overnight (approximately 18 hours). The excessfluorophore was separated from the labeled DNAs first by an ethanolprecipitation. The precipitated materials were resuspended in water andpassed over a Sephadex G-25 column that was 28 cm high with an internaldiameter of 1 cm. The desired fractions (the column void volumes) wereeluted with water and dried to reduce the total volumes. A secondethanol precipitation of the labeled DNAs completed the purification.Absorbance spectra of the labeled products showed that 5.5% of the totalnucleotides in the 5% (total nucleotides) aminated DNA preparation werelabeled. This procedure provides the CTMR probe.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were created to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei, Before hybridizing the slide was placed for 2 minutesin a denaturing solution of 70% formamide/0.3 M NaCl/30 mM sodiumcitrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85%, and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by heating to 70° C.for 5 minutes then allowed to hybridize at 37° C. for one hour. The mixwas applied directly to the slide, covered with a glass coverslip, andallowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 sodiumcitrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #7 or #8 for CTMR and set #1 or#2 for DAPI.

Qualitative Results for 5% aminated, 5.5% CTMR-labeled (based upon totalnucleotides) probe:

TABLE XI [Blocking DNA]; Visual Description [CTMR Probe] P = placental,(slide under microscope 40-100%) (ng/10 μl) C = Cotl (μg/10 μl)Specificity Intensity 316 2.25 P ++ +++ 1,000 2.25 P +++ +++ 316 6.75 P+++ ++ 1,000 6.75 P +++ ++++ 100 1.3 C  ++++ +++ 316 1.3 C  ++++ ++++1,000 1.3 C  ++++ ++++ 100 3.9 C  ++++ ++++ 316 3.9 C  ++++ ++++ 1,0003.9 C  ++++ ++++ Code: Specificity: (−) none apparent, (+) small amountof specificity, (++) reasonable specificity, (+++) good specificity,(++++) very good specificity. Intensity: (−) not visible, (+) barelyvisible, (++) fairly visible, (+++) bright, (++++) very bright

CTMR-labeled probes provided very good specificity and intensity, evenwith the 5% labeled (total nucleotides) probe. A probe prepared from the4.6% (based on deoxycytosines) aminated DNA similarly showed very goodresults.

EXAMPLE 8 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore5-(and-6)-carboxyfluorescein, Succinimidyl Ester(CFl)

Transaminated probe precursor DNA (average length 300 bp, 20% ofdeoxycytosines aminated) specific to human chromosome 4 were conjugatedwith 5-(and-6)-carboxyfluorescein, succinimidyl ester (CFl). Fiftymicrograms of transaminated DNA were dried and then resuspended in 377microliters of 200 mM MOPS, pH 7.4. Twenty-two and eight tenthsmicroliter of a 50 mM solution of CFl in N,N-dimethylformamide (a150-fold molar excess) was added to the transaminated DNA. This reactionproceeded with stirring in darkness at room temperature overnight(approximately 18 hours). The excess fluorophore was separated from thelabeled DNA first by an ethanol precipitation. The precipitated materialwas resuspended in water and passed over a Sephadex C-25 column that was28 cm high with an internal diameter of 1 cm. The desired fraction (thecolumn void volume) was eluted in water and dried to reduce the totalvolume. A second ethanol precipitation of the labeled DNA completed thepurification. An absorbance spectrum showed that 1.6% of the bases werelabeled. The procedure provides the CFl probe.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were treated to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei. Before hybridizing the slide was placed for 2 minutesin a denaturing solution of 70% formamide/0.3 M NaCl/30 mM sodiumcitrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85% and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe completed hybridization mixture were denatured by heating to 70° C.for 5 minutes and then allowed to hybridize at 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 H NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #4, #5, #6, #14 for CFl and set#1 or #2 for DAPI.

TABLE XII Qualitative Results [Blocking DNA]; Visual Description [CFIProbe] P = placental, (slide under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity   316 2.25P + ++   316 6.75P − +++1,000 6.75P − ++++   100 1.3C ++ ++   316 1.3C ++ +++ 1,000 1.3C − ++++  100 3.9C + +++   316 3.9C + +++ 1,000 3.9C ++ +++  100* 1.3C +++ +++ 316* 1.3C ++ +++ 1,000* 1.3C − ++ Code: Specificity: (−) none apparent,(+) small amount of specificity, (++) reasonable specificity, (+++) goodspecificity Intensity: (−) not visible, (+) barely visible, (++) fairlyvisible, (+++) bright, (++++) very bright *No carrier DNA present.

CFl-labeled probes provided good intensity with sufficient levels ofspecificity.

EXAMPLE 9 Labeling of Transaminated Chromosome-Specific-Probe PrecursorDNA with the Fluorophore Fluorescence-5-isothiocyanate (FITC)

Two different transaminated probe precursor DNAs (average length 300 bp,one to 20%, the other to 4.6% of deoxycytosines aminated) specific tohuman chromosome 7 were conjugated with fluorescein-5-isothiocyanate(FITC). Forty micrograms of each such transaminated DNA were dried andthen resuspended in 244 microliters of 50 mM sodium borate, pH 9.3. Sixand one tenth microliters of a 50 mM solution of FITC inN,N-dimethylformamide (a 50-fold molar excess) was added to thesuspensions of transaminated DNA. These reactions proceeded withstirring in darkness at room temperature overnight (approximately 18hours). The excess fluorophore was separated from the labeled DNAs firstby an ethanol precipitation. The precipitated materials were resuspendedin water and each passed over a Sephadex G-25 column that was 28 cm highwith an internal diameter of 1 cm. The desired fractions (the columnvoid volumes) were eluted with water and dried to reduce the totalvolumes. A second ethanol precipitation of the labeled DNAs completedthe purification. Absorbance spectra of the labeled products showed that2.2% of the total nucleotides in the 5% (total nucleotides) aminated DNApreparation were labeled and that 0.42% of the total nucleotides in the1.2% (total nucleotides) aminated DNA preparation were labeled.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were created to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei. Before hybridizing the slide was placed for 2 minutesin a denaturing solution of 70% formamide/0.3 M NaCl/30 mM sodiumcitrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85% and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier).

The concentrations of blocking DNA (Cot1 DNA or sonicated humanplacental DNA) and probe added to the basic mix were varied. Tenmicroliters of the complete hybridization mixture was denatured byheating to 70° C. for 5 minutes and then allowed to hybridize at 37° C.for one hour. The mix was applied directly to the slide, covered with aglass coverslip, and allowed to hybridize overnight at 37° C. in ahumidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #4, #5, #6 or #14 for FITC andset #1 or #2 for DAPI.

TABLE XIII Qualitative Results [Blocking DNA]; Visual Description [FITCProbe] P = placental (slide under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity 5% aminated, 2.2% FITC-labeled(based on total nucleotides) probe: 32 2.25P − + 102 2.25P + ++ 3232.25P − +++ 32 6.75P − − 100 6.75P − − 316 6.75P + ++ 1,000 6.75P + +++100 1.3C − ++ 316 1.3C + +++ 1,000 1.3C − ++++ 1.2% aminated, 0.42%FITC-labeled (based on total nucleotides) probe: 100 1.3C ++++ +++ 3161.3C ++++ ++++ Code: Specificity: (−) none apparent, (+) small amount ofspecificity, (++) reasonable specificity, (+++) good specificity, (++++)very good specificity. Intensity: (−) not visible, (+) barely visible,(++) fairly visible, (+++) bright, (++++) very bright

FITC-labeled probes provide very good specificity and intensity when thedegree of labeling is kept low. Specificity is poor when the degree oflabeling is high.

EXAMPLE 10 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore 7-Diethylaminocoumarin-3-carboxylic Acid,Succinimidyl Ester (DECCA)

Transaminated probe precursor DNA (average length 300 bp, 20% ofdeoxycytosines aminated) specific to human chromosome 4 were conjugatedwith 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester(DECCA). Forty micrograms of transaminated DNA were dried and thenresuspended in 364 microliters of 200 mM MOPS, pH 7.4. Thirty-six andfour tenths microliters of a 25 mM solution of DECCA inN,N-dimethylformamide (a 150-fold molar excess) was added to thetransaminated DNA. This reaction proceeded with stirring in darkness atroom temperature overnight (approximately 18 hours). The excessfluorophore was separated from the labeled DNA first by an ethanolprecipitation. The precipitated material was resuspended in water andpassed over a Sephadex C-25 column that was 28 cm high with an internaldiameter of 1 cm. The desired fraction (the column void volume) waseluted with water and dried to reduce the total volume. A second ethanolprecipitation of the labeled DNA completed the purification. Anabsorbance spectrum showed that 1.9% of the bases were labeled. Thisprocedure provides the DECCA probe.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were treated to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei. Before hybridizing the slide was placed for 2 minutesin a denaturing solution of 70% formamide/0.3 M NaCl/30 mM sodiumcitrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85% and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by heating to 70° C.for 5 minutes and then allowed to hybridize at 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the race of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 0.2 μg propidium iodide/ml as a generalchromosome stain to permit the visualization of all chromosomes. Theslide was then viewed with a fluorescence microscope using filter set #3for DECCA and set #10 for propidium iodide. Since the emissioncharacteristics of filter set #3 are poorly matched to the emissionmaximum of DECCA, the observed intensities are therefore lower thanexpected for a filter set better matched to the spectral properties ofDECCA such as filter set #13.

TABLE XIV Qualitative Results [Blocking DNA]; Visual Description (slide[DECCA Probe] P = placental, under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity 316 2.25P + + 1,000 2.25P + + 1001.3C − + 316 1.3C ++ ++ 1,000 1.3C +++ ++ Code: Specificity: (−) noneapparent, (+) small amount of specificity, (++) reasonable specificity,(+++) good specificity Intensity: (−) not visible, (+) barely visible,(++) fairly visible (+++) bright, (++++) bright

DECCA-labeled probes can provide good specificity, particularly whenusing Cot1 DNA, and reasonable intensity, even when using a relativelypoorly adapted filter set such as set #3. When later viewed with filterset #13, equivalent specificity and higher intensity were observed.

EXAMPLE 11 Labeling of Transaminated Chromosome-Specific Probe PrecursorDNA with the Fluorophore Tetramethylrhodamine-5-(and 6)-isothiocyanate(TRITC)

Transaminated probe precursor DNA average length 300 bp, 20% ofdeoxycytosines aminated) specific to human chromosome 7 were conjugatedwith tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC). Fortymicrograms of transaminated DNA were dried and then resuspended in 244microliters of 50 mM sodium borate, pH 9.3. Six and one tenth μl of a 50mM solution of TRITC in dimethyl sulfoxide (a 50-fold excess) was addedto the transaminated DNA. This reaction proceeded with stirring indarkness at room temperature overnight (approximately 18 hours). Theexcess fluorophore was separated from the labeled DNA first by anethanol precipitation. The precipitated material was resuspended inwater and passed over a Sephadex C-25 column that was 28 cm high with aninternal diameter of 1 cm. The desired fraction (the column void volume)was eluted with water and dried to reduce the total volume. A secondethanol precipitation of the labeled DNA completed the purification. Anabsorbance spectrum showed that 3.4% of the bases were labeled.

This embodiment of the invention was tested by in situ hybridizationusing conventional methods for the preparation of chromosome metaphasespreads. The target DNA consisted of cultured normal white blood cellsthat were created to arrest the cells in metaphase. These cells weredropped onto a glass microscope slide from about three feet to breakopen the nuclei. Before hybridizing the slide was placed for 2 minutesin a denaturing solution of 70% formamide/0.3 M NaCl/30 mM sodiumcitrate (pH 7) at 70° C. The slide was then dehydrated by passingthrough 70%, 85%, and 100% ethanol baths (2 minutes each).

The hybridization mix that was placed on slides (10 μl) was here always50% formamide/10% dextran sulfate/0.3 M NaCl/30 mM sodium citrate (pH7)/0.5 μg sonicated salmon sperm DNA (used as a carrier). Theconcentrations of blocking DNA (Cot1 DNA or sonicated human placentalDNA) and of probe added to the basic mix were varied. Ten microliters ofthe complete hybridization mixture were denatured by heating to 70° C.for 5 minutes and then allowed to hybridize at 37° C. for one hour. Themix was applied directly to the slide, covered with a glass coverslip,and allowed to hybridize overnight at 37° C. in a humidified chamber.

The next day the excess probe was removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly to the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The antifadesolution optionally contained 1.0 μg 4,6-diamidino-2-phenylindolehydrochloride (DAPI)/ml as a general chromosome stain to permit thevisualization of all chromosomes. The slide was then viewed with afluorescence microscope using filter set #7 and #8 for TRITC and set #1or #2 for DAPI.

TABLE XV Qualitative Results [Blocking DNA]; Visual Description [TRITCProbe] P = placental, slide under microscope 40-100%) (ng/10 μl) C =Cotl (μg/10 μl) Specificity Intensity 316 2.25P −/+ + 95 2.25P + + 3002.25P + ++ 32 6.75P + ++ 100 6.75P +++ ++/+++ 316 6.75P +++ +++ 1,0006.75P ++/+++ ++++ 100 1.3C +++ +++ 316 1.3C ++ +++ 1,000 1.3C + ++++Code: Specificity: (−) none apparent, (+) small amount of specific (++)reasonable specificity, (+++) good specificity Intensity: (−) notvisible, (+) barely visible, (++) fairly visible, (+++) bright, (++++)very bright

TRITC-labeled probes can provide both specificity and high intensityeven when using the relatively highly labeled (5% of total nucleotides)probes.

EXAMPLE 12 Use of Multiple Fluorescently Labeled Reagents

The target DNA consisted of cultured normal white blood cells that weretreated to arrest the cells in metaphase. These cells were dropped ontoa glass microscope slide from about 3 feet to break open the nuclei.Before hybridizing the slide was placed for 2 minutes in a denaturingsolution of 70% formamide/0.3 M NaCl/30 mM sodium citrate (pH 7) at 70°C. The slide was then dehydrated by passing through 70%, 85%, and 100%ethanol baths (2 minutes each).

The hybridization mix that was placed on each slide (10 μl) containedtwo to five fluorophore-labeled probes, specific for differentchromosome pairs, and 50% formamide/10% dextran sulfate/0.3 M NaCl/30 nMsodium citrate (pH 7). In some experiments the mix contained 0.5 μgsonicated salmon sperm DNA (used as carrier). The concentrations ofblocking DNA (Cot1 DNA or sonicated human placental DNA) added to thebasic mix were varied. Ten microliters of the complete hybridizationmixture were denatured by heating to 70° C. for 5 minutes and thenallowed to hybridize at 37° C. for one hour. This mix was applieddirectly to the slide, covered with a glass coverslip, and allowed tohybridize overnight at 37° C. in a humidified chamber.

The next day the excess probes were removed by washing the slide threetimes, 15 minutes each, at 45° C. in 50% formamide/0.3 M NaCl/30 mMsodium citrate (pH 7), then washing the slide in 0.3 M NaCl/30 mM sodiumcitrate (pH 7) for 15 minutes at 45° C., followed by washing the slidein 0.1 M sodium phosphate (pH 7)/0.1% NP40 detergent (NP40 isoctylphenoxypolyethoxyethanol, which is a nonionic surfactant sold byCalbiochem, La Jolla, Calif.) for 15 minutes at 45° C. Finally the slidewas washed twice, for two minutes each, in 0.1 M sodium phosphate (pH7)/0.1% NP40 detergent at room temperature and the slide air dried. 7.5μl of antifade solution, used to reduce the rate of fluorophorephoto-oxidation, was applied directly co the slide and a coverslipplaced over the drop of antifade solution. (See Example 3). The slidewas then viewed with a fluorescence microscope using various filter setsto differentiate the emission from the several different fluorescentlabels.

The combinations and concentrations of labeled probes usedsimultaneously in hybridizations are listed in the following table. Eachcombination of two, three, four, or five probes which was tested islisted together in the table without spacing. Some filter sets allowedonly one fluorophore to be visualized while others allowed twofluorophores to be observed simultaneously. For example, the DECCAfilter set (#3) allows the observation of both DECCA- and CF1-labeledprobes. The DECCA- and CF1-stained chromosomes can be distinguished,though, because the CF1 filter set (#5, #6 or #14) allows visualizationof only the CF1-stained chromosomes. This then identifies which of thetwo stained chromosome pairs results from fluorescein and which resultsfrom DECCA-labeled probes when viewed using the DECCA filter set #3.Likewise, the filters initially used were not able to distinguishtetramethylrhodamines from the rhodamine 101 derivatives, Texas Red andCXR, individually. However, using the TRITC filter set #8 the CTMRlabels appear yellow-orange while the CXR and Texas Red labels appearmore distinctly red allowing a visual distinction between the twodifferent stains.

TABLE XVI BLOCKER CARRIER PROBES DNA DNA Human Conc. P = placental DNAChromosome Label (ng/10 μl) C = Cotl DNA 2 Probes, SimultaneousHybridizations: 4 CTMR 316 6.75P 0.5 1 AMCA 1000 4 CTMR 316 13.5P 0.5 1AMCA 1000 3 Probes, Simultaneous Hybridizations: 1 CTMR 316 1.3C 0.5 4AMCA 316 8 DECCA 316 1 CTMR 316 3.9C 0.5 4 AMCA 316 8 DECCA 316 4Probes, Simultaneous Hybridizations: 1 CTMR 316 1.3C 0 4 AMCA 316 7 CXR316 8 DECCA 316 1 CTMR 316 1.3C 0 4 CFl 316 6 AMCA 1000 8 DECCA 316 1AMCA 316 1.3C 0 4 CXR 316 6 CTMR 316 8 DECCA 316 1 CTMR 316 1.3C 0 4 CXR316 6 AMCA 1000 8 DECCA 316 5 Probes, Simultaneous Hybridizations: 1AMCA 316 1.3C 0 4 CFl 316 6 CTMR 316 7 CXR 316 8 DECCA 316

Each multiple hybridization listed in the table provided similarresults. Visual examination of the stained metaphase spreads showed thateach labeled probe specifically stained one and only one pair ofchromosomes. In addition, each specifically stained pair of chromosomeswas distinguishable from the others based upon the color of the lightemitted by the stain. Using filter sets #2, #3 (or preferably #13), #5,(or #6), #8, and #9, it was found possible to visualize each of thepairs of target chromosomes from the others.

EXAMPLE 13 Labeling of Chromosome-Specific DNA Probe Precursors withVarious Fluorophores

Transaminated of DNA probe precursors prepared as described in Examples1 and 2 (each respective one of such probe segments having an averagelength of 300 bp with from 1 to 5% of the total nucleotides thereofaminated at deoxycytidine nucleotides) and each of such probe segmentsbeing specific to an individual human chromosome, were each conjugatedwith one of several amine-reactive fluorophores.

The conjugation reaction conditions varied according to whichamino-reactive functionality was present on the fluorophores, thereaction buffer 50 mM (millimolar) sodium borate, pH 9.3. ForN-hydroxysuccinimide ester derivatives of fluorophors, the reactionbuffer was 200 mM MOPS, pH7.4. 30 to 100 μg DNA/ml for forN-hydroxysuccinimide ester reactions. THe final amine-reactivefluorophore concentrations were 1.2 mM for isothiocyanate derivativesand 2.3 mM for N-hydroxysuccinimide esters. The mM solutions of theamine-reactive fluorophores were prepared by dissolving the fluorophorein a non-reactive solubilizing solvent, such as dimethylformamide (DMF)or dimethylsulfoxide (DMSO). The reaction conditions are summarized inTable XVII below.

EXAMPLE 13 Labeling of Chromosome-Specific DNA Probe Precursors withVarious Fluorophores

Transaminated DNA probe segments prepared as described in Examples 1 and2 (each respective one of such probe segments having an average lengthof 300 bp with from 1 to 5% of the total nucleotides thereof aminated atdeoxycytidine nucleotides) and each of such probe segments beingspecific to an individual human chromosome, were each conjugated withone of several amine-reactive fluorophores.

The conjugation reaction conditions varied according to whichamino-reactive functionality was present on the fluorophores. Forisothiocyanate derivatives of fluorophores, the reaction buffer 50 mMsodium borate, pH 9.3. For N-hydroxysuccinimide ester derivatives offluorophores, the reaction buffer was 200 mM MOPS, pH 7.4. 30 to 100 μgof aminated DNA was dissolved in the reaction buffer and a 20 mMsolution of the reactive fluorophore added. The resulting reactionmixture was then mixed continuously overnight, in the dark, at roomtemperature.

The final DNA concentration in the reaction mixture was 160 μg DNA/mlfor isothiocyanate reactions and 100 μg DNA/ml for N-hydroxy-succinimideester reactions. The final amine-reactive fluorophore concentrationswere 1.2 mM for isothiocyanate derivatives and 2.3 mM forN-hydroxysuccinimide esters. The 20 mM solutions of the amine-reactivefluorophores were prepared by dissolving the fluorophore in anon-reactive solubilizing solvent, such as dimethylformamide (DMF) ordimethylsulfoxide (DMSO). The reaction conditions are summarized inTable XVII below.

TABLE XVII LABELING REACTION CONDITIONS [FLUORO- PHORE]^(b) [DNA]FLUOROPHORE SOLVENT^(a) BUFFER (mM) (μg/ml) ISOTHIO- 50 mM 1.2 160CYANATES Borate, pH 9.3 EITC DMF ErITC DMF N-HYDROXY- 0.2 M MOPS, 2.3100 SUCCINIMIDYL pH 7.4 ESTERS HCCA DMF FCHA DMSO DMBP DMF FAP DMSOOTHER CBAA DMSO 0.1 M 2.3 100 NaHCO₃, pH 8.4 ^(a)Fluorophore isdissolved in the specified solvent at a concentration of 20 mM prior toadding the fluorophore stock to the reaction buffer containing theaminated DNA. ^(b)Based on 20% amination of dN's, the molar ratio offluorophore-to-aminated dC is 50 for isothiocyanate reactions and 150for sulfonic acid chloride and N-hydroxysuccinimide ester reactions.

The labeled probes were purified by ethanol precipitations of the DNAand gel permeation chromatography as follows. Labeled DNA in thereaction mixtures was precipitated by adding 1/10th volume of 2.6 Msodium acetate, pH 5.4, and 2.5×volume of ethanol. The resultingsolution was then equilibrated in either a −20° C. freezer overnight orin dry ice for 15 minutes before centrifuging the solution at 7,800×gfor 10 minutes. The supernatant was discarded and the labeled DNA pelletdried and resuspended in about 300 μl of water. The DNA solution wasthen applied to a Sephadex G-25 column (1 cm diam.×28 cm height), whichwas pre-equilibrated with water, and the purified labeled DNA collectedin the column void volume upon elution with water. The labeled DNA wasthen precipitated a second time with ethanol, either directly from thecolumn solution collected from the column or following volume reductionin a centrifugal evaporator. The precipitated purified labeled DNA wasthen dissolved in about 300 μl of water and stored frozen at −20° C.

The Cascade Blue acetylazide CBAA was reacted with DNA and purified asdescribed above for N-hydroxysuccinimide esters except that the reactionbuffer was 0.1 M NaHCO₃, pH 8.4, as recommended by the manufacturer.

The number of labels attached per nucleotide for each probe wasdetermined by the same procedure as employed in the preceding Examples3-11. Thus, the percentage of nucleotides aminated within a probe wasdetermined prior to fluorophore attachment by the enzymaticdigestion/FPLC chromatography procedure described hereinabove. Thepercentage of nucleotides attached to a fluorophore after labeling wasdetermined from the absorbance spectrum using the absorbance at 260 nmand the absorbance at the long wavelength absorbance maximum of thefluorophore. The ratio of the absorbance at 260 nm to the absorbance atthe long wavelength absorbance maximum of the unconjugated dye wasmultiplied by the long wavelength absorbance of the conjugate and theproduct subtracted from the absorbance at 260 nm of the conjugate tocalculate the absorbance contribution at 260 nm due to DNA. Thisadjusted absorbance value at 260 nm was divided by an absorbanceextinction coefficient of 10,000 M⁻¹ cm⁻¹ nucleotide⁻¹ to obtain thenucleotide concentration of probe in the solution. The long wavelengthabsorbance maximum of the conjugate was divided by the extinctioncoefficient of the fluorophore to obtain the fluorophore concentrationin conjugate. The percentage of nucleotides attached to fluorophore inthe probe was calculated as the ratio of the fluorophore concentrationto nucleotide concentration multiplied by 100. There is some presentuncertainty in the values of the extinction coefficients of thefluorophore and the DNA, as well as the absorbance ratio of theunconjugated fluorophore, since the extinction coefficients of thefluorophores may be altered by conjugation to the DNA. Also, thecalculated labeling percentages are sometimes higher than the percentageamination due to the fact that the probe DNA may not be completely freefrom unconjugated label. This results either when the label binds to theDNA noncovalently or when the dye forms aggregates which are excluded bythe Sephadex G25 columns and precipitate in the ethanol solutions withthe DNA. The presence of unconjugated label in the probe preparationdoes not necessarily mean that a probe will perform poorly in in situhybridizations.

TABLE XVIII QUALITATIVE RESULTS OF HYBRIDIZATIONS USING WHOLE CHROMOSOMEPAINTING PROBES. VISUAL DESCRIPTION⁵ FLUOROPHORE- % % [PROBE]⁴ (slideunder micropscope-40X) Filter Set PROBE¹ AMINATION² LABELING³ (ng/10 μl)Specificity Intensity Ref #⁶ HCCA-C4 0.90 0.48 320 +++ ++ 12 HCCA-C4 2.90.90 320 +++ +++ 12 EITC-C4 0.90 0.90 320 +++ ++++ 15 EITC-C4 2.9 1.44320 ++ ++++ 15 ErITC-C4 0.90 0.87 320 ++ ++ 15 ErITC-C4 2.9 1.02 320 +++ 15 FCHA-C4 0.90 0.74 160 +++ +++ 5, 6, 14 FCHA-C4 2.9 2.01 160 +++++++ 5, 6, 14 DMBP-C4 0.9 0.56 320 ++ ++/+++ 5, 6, 14 CBAA-C4 2.9 1.41316 ++ + 11 FAP-C4 1.0 0.57 160 +++ ++++ 5, 6, 14 FAP-C4 3.0 0.94 160 ++++++ 5, 6, 14 ¹Labeled probes are indicated as the fluorophore name orabbreviation followed by the number of the chromosome specificallystained by the whole chromosome paint probe (e.g. C4 = chromosome 4).²The percentage of the total nucleotides which contain aliphatic aminelinker groups, as determined by enzymatic digestion and FPLCfractionation of nucleosides. ³The percentage of the total nucleotideswhich contain covalently attached fluorophores, as determined byabsorbance spectroscopy. ⁴The concentration of labeled probe in thehybridization solution. ⁵Specificity is indicated as follows: (−) = noneapparent, (+) = small amount of specificity, (++) = reasonablespecificity, (+++) = good specificity, and (++++) = very goodspecificity. Intensity is indicated as: (−) = not visible, (+) = barelyvisible, (++) = fairly visible, (+++) = bright, and (++++) = verybright. ⁶See Table V.

In situ hybridizations of whole chromosome painting probes wereperformed as described in Examples 3-11. Filter sets used to view thefluorescently stained chromosomes are listed in Table V.

Table XVIII presents some qualitative results of in situ hybridizationsusing the fluorophores described in this example and shown in Table XVIIwhich are in probes that specifically bind to human chromosome #4. Theseresults show that all of these fluorescent labels provide labeled probeswhich are visually detectable under the microscope and show specificityfor staining chromosome #4. The results in Table XVIII also show thatthe intensity and the specificity of labeled chromosome #4 probestaining is dependent upon the level of probe amination (and thereforelabeling). HCCA- and FCHA-labeled probes display similar specificity butincreased intensity when the probe is aminated at a level of 3% relativeto 1%. EITC-, FAP- and ErITC-labeled probes display similar intensity atboth amination levels, but decreased specificity at the higher aminationlevel. For some labels, the optimum amination level is also dependentupon the chromosome library DNA. For example, FAP-labeled chromosome #1and #7 specific probes performed better with 3% aminated probes thanwith 1% aminated probes, in contrast to the chromosome #4 specific probewhich performed better at 1% amination (results not shown in table).Some variation in the specificity or clarity of green fluorophoresstains such as FAP, FCHA and CFL has been observed which variation isgreater than that which is observed with, for example, with orangefluorophores such as CTMR.

Quantitative analyses of stained metaphase spreads were performed inorder to look more closely at the dependence of probe staining onamination level for some of the labeled probes. The results arepresented in Table XIX.

TABLE XIX QUANTITATIVE PERFORMANCE OF DIFFERENT LABLES AT DIFFERENTLEVELS OF PROBE AMINATION. CHROMOSOME 12 CHROMOSOME 4 % Amination %Amination LABEL 0.86 2.0 2.8 6.9 0.9 2.9 4.8 HCCA % Labeling 0.86 0.671.71 2.14 0.48 0.90 1.06 Spec Intensity 74.9 76.6 105 48.1 128 150 211Nonspec Intensity 43.7 42.5 50.2 36.2 52.1 59.9 65.2 Bkg Intensity 36.735.2 38.3 33.9 39.3 41.6 43.0 Gross Spec/Nonspec 1.72 1.80 2.09 1.332.46 2.50 3.24 Net Spec/Nonspec 5.46 5.67 5.57 6.13 6.97 5.91 7.57 CBAA% Labeling 1.41 2.29 Spec Intensity 301 305 Nonspec Intensity 253 264Bkg Intensity 243 252 Gross Spec/Nonspec 1.19 1.16 Net Spec/Nonspec 6.134.36 ErITC % Labeling 0.87 1.02 Spec Intensity 44.4 41.9 NonspecIntensity 33.5 31.8 Bkg Intensity 26.8 25.6 Gross Spec/Nonspec 1.32 1.32Net Spec/Nonspec 2.61 2.65 EITC % Labeling 0.57 0.45 0.54 0.88 0.90 1.441.26 Spec Intensity 122 81.6 64.9 182 227 436 135 Nonspec Intensity 73.754.7 48.8 128 80.6 178 99.6 Bkg Intensity 56.7 47.6 43.9 84.6 52.4 99.167.3 Gross Spec/Nonspec 1.66 1.49 1.33 1.42 2.81 2.45 1.36 NetSpec/Nonspec 3.85 4.81 4.30 2.24 6.17 3.46 2.11 FCHA % Labeling 0.661.30 1.75 3.14 0.74 2.01 3.63 Spec Intensity 170 307 345 382 269 760 437Nonspec Intensity 95.3 156 113 248 128 208 152 Bkg Intensity 57.8 7.4562.1 153 70.4 108 94.6 Gross Spec/Nonspec 1.78 1.97 3.05 1.54 2.09 3.662.87 Net Spec/Nonspec 2.99 2.85 5.55 2.41 3.42 6.54 5.95 DMBP % Labeling0.38 0.71 1.01 0.56 0.56 1.51 2.14 Spec Intensity 31.2 48.2 48.3 60.739.2 87.1 101 Nonspec Intensity 23.8 26.7 27.6 32.7 27.2 35.9 41.3 BkgIntensity 21.8 22.3 23.3 25.6 24.1 26.6 29.8 Gross Spec/Nonspec 1.311.80 1.75 1.86 1.44 2.42 2.45 Net Spec/Nonspec 4.84 5.88 5.70 4.94 4.916.48 6.24

Analyses were performed by first recording a digital image of afluorescently-stained metaphase spread using a cooled CCD camera(Photometrics, Tucson, Ariz., Series 200 camera) interfaced to aMacintosh II fx computer. Image processing software (IP Labs, SignalAnalytics, Vienna, Va.) was used to separately determine the averageimage pixel intensity of the specifically-stained chromosomes (SpecIntensity), the remaining chromosomes (Nonspec Intensity), and thenon-chromosome region (Bkg Intensity). Specificity is considered to bethe ratio of the specifically stained chromosome intensity-to-thenonspecifically-stained chromosome intensity, either before (GrossSpec/Nonspec) or after (Net Spec/Nonspec) subtraction of the backgroundintensity.

The intensities and specificities (intensity ratios) listed in Table XIXshow several significant trends. In general, the specific stainintensities increase with increasing probe amination level, and hencepercent labeling. Often the increase in intensity with amination levelreaches a maximum value and decreases with further increase in aminationlevel. The specificities also show a similar behavior. Ideally, theintensity and specificity would be maximal at the same amination leveland this would present the optimal amination level for that particularprobe DNA and label. This is not always the case, however, and theoptimal amination level must be selected as a trade off betweenintensity and specificity. Another factor that affects these values isprobe concentration. At lower concentrations, the specificity usuallyimproves while the intensity decreases. A probe which provides a highintensity stain and a low specificity can benefit by lowering the probeconcentration to improve the specificity. If the intensity of the probestaining is already low, then lowering the probe concentration mayimprove the specificity, but reduces the staining intensity to anunacceptable level.

The numbers listed in Table XIX reflect only average intensities withina metaphase region. Variations of intensity along a chromosome oroutside of the chromosomes are lost in these calculations. Therefore,specificity can actually be lower than indicated by the averages. Forexample, a labeled-chromosome #6 probe might stain small regions ofchromosomes other than #6 as intensely as it stains chromosome #6. Thiscould be a commercially unacceptable specificity, however, the averageintensities of the non-specifically stained chromosomes would still below due to averaging the bright “spots” over the whole chromosomes, andthe lack of specificity would be missed. Therefore, visual observationsof background and chromosome staining must be considered with thedigital information. Considering the digital and visual information, theoptimal amination levels for the various probes are now believed to beabout 3% for HCCA, CBAA, FCHA, and DMBP, and about 1% for EITC and ErITCbased on available data. These are very general numbers since somechromosome-to-chromosome variation is observed in the optimal aminationlevels and these analyses are based upon only the laboratory workdescribed here.

EXAMPLE 14 Direct Label Probe Composition Specifically Complementary tothe Centromere Region of Human Chromosome #12

A cloned DNA sequence known to be specifically complementary to thecentromere region of human chromosome #12 was prepared by the proceduredescribed in Bittner et al. U.S. Pat. Ser. No. 07/762,912 published asPCT Patent Application WO 93/0246, on Apr. 1, 1993, filed on even dateherewith, (identified as purified plasmid DNA cloned sequence #1-1, andhaving a 3.5 k bp insert, and known to contain DNA repeated sequences).This sequence was disrupted into small fragments of approximately 300base pairs by sonication using a Branson Sonifier 450 (Danbury, Conn.).DNA from the plasmid preparation was sonicated in 2 mls of water at aconcentration of 500 μg/ml. The solution was contained in a 5 mlpolypropylene tube which was immersed in a dry ice/ethanol bath toprevent boiling during sonication. The microtip of the sonication devicewas immersed in this solution until the tip was 2-5 mm from the bottomof the tube. Sonication was carried out at an output power of 25-30watts, discontinuously, with an 80% duty cycle (on 80% of the time, off20% of the time), for a period of 5-7 minutes. Following sonication, theDNA was precipitated by the addition of 0.2 ml of 3M sodium acetate (pH5.5) and 4.5 mls of ethanol. The precipitate was recovered bycentrifugation for 5 minutes at 8,000×g and vacuum dried.

To prepare bisulfite buffer, 1.7 ml of concentrated HCl was slowly addedto 1 ml of deionized H₂O on ice. 1 ml fresh ethylene diamine (Sigma Cat.#E-4379) was then slowly added on ice. After dissolution of the ethylenediamine, the solution was warmed to room temperature and 0.475 g sodiummetabisulfite (Aldrich Cat. #25, 555-6) was added. Concentrated HCl wasthen slowly added to the bisulfite mixture until the pH reached 7.0 andthe volume of the solution was adjusted to 5.0 ml.

To transaminate probe DNA, 1 mg of sonicated DNA was resuspended in 500μl of water. The DNA was denatured by boiling at 100° C. for 5 minutesthen quickly chilled in an ice water bath. The transamination reactionwas initiated by the addition of 4.5 ml of bisulfite buffer. Reaction inbisulfite buffer was allowed to proceed for 2 days at 37° C. The DNAsolution was desalted by routine dialysis against 5-10 mM sodium boratebuffer (pH 8.0). After dialysis, 0.6 ml of 3M sodium acetate (pH 5.5)was added. The aminated DNA was precipitated with 2 volumes of ethanoland recovered after centrifugation at 8,000×g for 10 minutes. Thepellets were vacuum dried and resuspended in 1 ml of water.

The extent of transamination of deoxycytidine was determined byenzymatic digestion of the aminated DNA followed by separation of theresulting nucleotides on an FPLC chromatography system as described inExample 2. This analysis indicated that 3.4% of total nucleotides or13.6% of deoxycytidines were transaminated.

Forty micrograms of aminated DNA sequence 1-1 derived from humanchromosome #12 as described in the afore referenced Bittner, et al.application Ser. No. 07/762,912 filed on even date herewith, publishedas PCT Patent Application WO 93/0246, on Apr. 1, 1993, was dried into a2 ml tube then resuspended in 362 μl 0.20 M MOPS(3-[N-Morpholino]propanesulfonic acid), pH 7.4. The fluorescent compound5- (and-6) carboxytetramethylrhodamine (CTMR), succinimidyl ester wasdissolved in dimethylformamide to 30 mm. A 150-fold molar excess of thefluorophore relative to the intermediate transaminated nucleotides(assuming 5% of the total nucleotides are transaminated) was added tothe DNA, in this case 37.9 μl of 30 mM CTMR. This labeling reactionproceeded in darkness at room temperature with the tube rotatingovernight.

The purification of the labeled probe away from the excess fluorophorewas a three step procedure. The first step was an ethanol precipitation.Any remaining ethanol was evaporated from the precipitated pellet, thenthe probe was resuspended in 300 μl water. This solution was passed overa Sephadex G-25 column 28 cm high and 1 cm diameter. The desiredfraction (the column void volume) was eluted with water and dried toreduce the total volume. A second ethanol precipitation completed thepurification and the dried pellet was resuspended in 300 μl water. Anabsorbance spectrum of the labeled probe showed 3.3% of the totalnucleotides were labeled.

EXAMPLE 15 Detection of the Centromere Region of Chromosome #12 by InSitu Hybridization

The probe composition of the preceding Example 14 was used to identify(detect) the centromere region of human chromosome #12 as follows:

The target DNA consisted of cultured normal white blood cells that weretreated to arrest the cells in metaphase. These cells were dropped ontoa microscope slide from a distance of about 2 to 3 feet to break openthe nuclei and expose the chromosomes. Unbroken or interphase cells werealso present on the slide surface. Before hybridizing, the slide wasplaced in a denaturing solution consisting of 70% formamide/0.3 MNaCl/30mM sodium citrate, pH 7.0, for 2 minutes at 70° C. The slide was thendehydrated by passage through 70%, 85%, and 100% ethanol baths (2minutes each). The slide was then warmed to approximately 40° C.

The hybridization mix that was placed on each slide was always 55%formamide/10% dextran sulfate/0.15 M NaCl/15 mM sodium citrate, pH 7.0.The concentrations of probe added to the basic hybridization mix variedto determine the optimal concentration needed to obtain acceptablesignal intensity and specificity. The reaction mixture also contained4.5 μg of sonicated human placental DNA added as carrier and blockingDNA. In addition to the unlabeled human placental DNA, approximately 96ng of fluorescein labeled, sonicated human placental DNA was added as agenomic counterstain. The preparation of such genomic counterstain istaught in copending Morrison et al. U.S. Ser. No. 07/762,928 filed oneven date herewith, published as PCT Patent Application WO 93/0245 onApr. 1, 1993. Ten μl of the completed hybridization mixture wasdenatured by heating at 70° C. for from 5 to 15 minutes and thenincubated at 37° C. for 5 minutes. The mix was applied directly to theslide, covered with a glass coverslip whose edges were sealed withrubber cement, and allowed to hybridize overnight at 42° C. in ahumidified chamber.

The next day the unbound probe was removed by washing the slide (threetimes, each for 15 minutes at 45° C.) in 0.3 MNaCl/30 mM sodium citrate50% formamide v/v, pH 7.0. A single wash (15. minutes at 45° C.) in 0.3M NaCl/30 mM sodium citrate (2×SCC, pH 7.0), followed. The final wash(15 minutes at 45° C.) was in 0.1 M sodium phosphate/0.1% NP40 detergent(PN buffer). Finally, the slide was washed twice in PN buffer (2 minutesat room temperature), and air dried. Ten microliters of antifade wasplaced over the target cells and a coverslip was placed over that. Theslides were viewed with a fluorescence microscope.

Results are tabulated in Table XX below:

TABLE XX Qualitative Results Hybridization Conditions Visual DescriptionCTMR Concentration (1) (2) Probe (3) (ng/10 μl) Intensity Specificity#1-1 33 ++++ ++++ 100 ++++ ++++ (1) Intensity: (−) not visible, (+)barely visible, (++) fairly visible, (+++) bright, (++++) very bright,(NE) cannot be evaluated (2) specificity: (−) none apparent, (+) lowspecificity, (++) reasonable specificity, (++++) good specificity, (NE)cannot be evaluated (3) Blocking DNA present (4.5 μg human placentalDNA/10 μl)

On the basis of the above results it is concluded that direct attachmentof the flurophore to the alphoid DNA probe of Example 14 produces aprobe composition which can readily be utilized for in situhybridization analysis of specific centromeres.

EXAMPLE 16 Direct Label Probe Composition Specifically Complementary tothe Centromere Region of Human Chromosome #8

A cloned DNA sequence known to be specifically complementary to thecentromere region of human chromosome #8 was prepared by the proceduredescribed in afore described Bittner et al. U.S. Ser. No. 07/762,912filed on even date herewith published as PCT Patent Application WO93/0246, on Apr. 1, 1993 (identified as purified plasmid DNA clonedsequence 10-4).

The DNA sequence from plasmid 10-4 was prepared by fermentation.Bacteria containing the plasmid 10-4 were streaked onto a YT agar platecontaining 200 μg/ml of ampicillin. A single colony from this plate wastransferred into 2 ml of 2 YT broth and allowed to grow overnight at 30°C. with agitation. This bacterial suspension served as the seed stockfor the fermentation process described in such Bittner et al. U.S. Ser.No. 07/762,912, published as PCT Patent Application WO 93/0246, on Apr.1, 1993 and the fermented culture and extraction of the DNA sequencewere as described therein. Fermentation yielded a cell mass of 204grams. Extraction of 122 grams of this pellet yielded 60 milligrams ofplasmid DNA. 1 milligram of the resulting plasmid 10-4 DNA sequence wassonicated as described in such Bittner et al. U.S. Ser. No. 07/762,912,published as PCT Patent Application WO 93/0246, on Apr. 1, 1993. Onemilligram of this sonicated DNA was resuspended in 1 ml of distilledwater. The DNA was denatured by boiling for 5 minutes and quicklycooling on ice.

9 mls of bisulfite buffer were added and a transamination reaction asdescribed in Example 14 was allowed to proceed for 2 days at 37° C. DNAproduct which resulted was desalted by routine dialysis against 10 mMsodium borate (pH 8.0). This resulting DNA was precipitated by theaddition of 0.1 volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes ofethanol, and the precipitated DNA was resuspended in water at aconcentration of 1 mg/ml. Forty micrograms of the resulting aminated DNAsequence 10-4 to the centromere of chromosome #8 was dried into a 2 mltube and then resuspended in 362 μl 0.20 M MOPS(3-[N-Morpholino]propanesulfonic acid), pH 7.4. The fluorescent compound5- (and-6) carboxytetramethylrhodamine (CTMR), succinimidyl ester wasdissolved in dimethylformamide to 30 mM. A 150-fold molar excess of thisfluorophore was added to the aminated DNA, in this case 37.9 μl of 30 mMCTMR. This labeling reaction proceeded in darkness at room temperaturewith the tube rotating overnight.

The purification of the labeled probe away from the excess fluorophorewas a subsequent three step procedure. The first step was an ethanolprecipitation. Any remaining ethanol was evaporated from theprecipitated pellet, then the probe was resuspended in 300 μl water.This solution was passed over a Sephadex G-25 column 28 cm high and 1 cmin diameter. The desired fraction (the column void volume) was elutedwith water and dried to reduce the total volume. A second ethanolprecipitation completed the purification and the dried pellet wasresuspended in 300 μl water. An absorbance spectrum of the resultingdirect label probe composition showed 3.1% of the total nucleotides werelabeled.

EXAMPLE 17 Detection of the Centromere Region of Chromosome #8 by InSitu Hybridization

The probe composition of the preceding Example 16 was used to identify(detect) the centromere region of human Chromosome #8 as follows:

16 ng of the direct label probe composition of Example 16 above wasdried into a 0.5 ml tube with a tight-fitting cap. The probe wasresuspended in 10 μl of 55% formamide/10% dextran sulfate/0.15M NaCl/15mM sodium citrate, pH 7.0, with 4.5 μg sonicated human placental DNAbeing added as blocker. This hybridization mixture was denatured byplacing the tube in a 70° C. water bath for 5 minutes.

A target slide which was prepared as described in Example 15 above wasdenatured for 3 minutes in a 70° C. solution of 70% formamide/2×SSC andthen dehydrated by passing successively through 70%, 85%, and 100%ethanol baths (2 minutes each). A drop of the thus previously de-naturedhybridization mixture was pipetted onto the slide and the drop wascovered with a coverslip. The coverslip was sealed onto the slide withrubber cement. The hybridization was allowed to proceed overnight in adark, humidified 37° C. chamber.

The next day the residual unbound probe was removed by washing the slide(three times, each for 15 minutes at 45° C.) in 50%formamide/0.3MNaCl/30 mM sodium citrate, pH 7.0. A single wash (15minutes at 45° C.) in 0.3M NaCl/30 mM sodium citrate (2×SSC, pH 7.0),followed. The slide was next washed in 0.1 M sodium phosphate/0.1% NP40detergent (PN buffer) (15 minutes at 45° C.). Finally, the slide waswashed twice in PN buffer (2 minutes at room temperature), and airdried. 7.5 μl of 1 μg/ml DAPI in an antifade solution was placed overthe target cells and a coverslip was placed over that.

The results obtained were tabulated as follows in Table XXI:

The technique and advantages of employing a chaotrope in thetransamination are taught in the aforereferenced Bittner et al. U.S.Ser. No. 07/762,912 published as PCT Patent Application WO 93/0246, onApr. 1, 1993, filed on even date herewith.

TABLE XXI Results of In Situ Hybridization with Fluorophore Direct LabelProbe Hybridization Conditions Visual Description CTMR (3) Concentration(1) (2) Probe (ng/10 μl) Intensity Specificity #10-4 16 ++++ ++++ (9 kbpinsert) Table XXI footnotes: (1) Intensity: (−) not visible, (+) barelyvisible, (++) fairly visible, (+++) bright, (++++) very bright, (NE)cannot be evaluated (2) Specificity: (−) none apparent, (+) lowspecificity, (++) reasonable specificity, (++++) good specificity, (NE)cannot be evaluated (3) * blocking DNA present (4.5 μg human placentalDNA/10 μl) Based on the above indicated results, it was concluded thatthe so produced direct label probe composition is well suited for use inin situ hybridization enumerations of specific chromosome centromeresusing fluoroscopic analysis. Other and further embodiments will beapparent in the art from the preceding description and examples. Nounreasonable limitations or the like to be drawn therefrom.

What is claimed is:
 1. A method of simultaneous detection of at leasttwo preselected target chromosomes or target chromosome regions by insitu hybridization using multiple direct label DNA probes comprising:(a) providing a plurality of at least two direct label DNA probescompositions, wherein each of the direct label DNA probe compositions:(i) comprise direct label DNA segments which are complementary to asingle target chromosome or target chromosome region and havefluorescent labels directly linked to the DNA segments, and (ii) isdetectable by use of a different fluorescent color, each color beingdetectable in the presence of other direct label probe compositions; (b)contacting the plurality of direct label DNA probe compositions underhybridizing conditions in in situ hybridization with a plurality oftarget chromosomes or target chromosome regions contained in a targetspecimen; and (c) detecting the target chromosomes or target chromosomeregions by identification of two or more fluorescent colors using adigital image of the target specimen.
 2. The method of claim 1 whichfurther comprises adding an excess of blocking DNA segments to themultiple probe composition.
 3. The method of claim 1 wherein the directlabel DNA segments comprise fluorescent labels covalently linked to thedirect label DNA segments via transaminated cytosine sites.
 4. Themethod of claim 1 wherein the target specimen comprises at least onehuman interphase cell.
 5. The method of claim 1 wherein the targetchromosomes or target regions of a chromosome are contained in humanamniotic cells.
 6. The method of claim 1 wherein the target chromosomesor target regions of a chromosome are extracted from tissue.
 7. Themethod of claim 1 wherein said fluorescent labels are each derivativesof a compound selected from the group consisting of7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester; Texas Redsulfonyl chloride; 5- (and 6-)-carboxy-X-rhodamine, succinimidyl ester;Lissamine rhodamine B sulfonyl chloride; 5- (and 6-)-carboxyfluorescein,succinimidyl ester; fluorescein-5-isothiocyanate;7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester;tetramethylrhodamine 5- (and 6-)isothiocyanate; 5- (and6-)carboxytetramethylrhodamine, succinimidyl ester;7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester; and 6-hexanoicacid, succinimidyl ester.
 8. The method of claim 1 wherein at least fourpreselected target chromosome regions are detected.
 9. The method ofclaim 1 wherein at least five preselected target chromosome regions aredetected.
 10. The method of claim 1 wherein a digital image is madeusing a CCD camera.
 11. The method of claim 1 further comprisingdetermining pixel intensity of the fluorescent colors.
 12. The method ofclaim 11 wherein the target specimen is human tissue.
 13. The method ofclaim 11 wherein at least four preselected target chromosome regions aredetected.
 14. The method of claim 11 wherein the direct label DNAsegments comprise fluorescent labels covalently linked to the directlabel DNA segments via transaminated cytosine sites.
 15. The process ofclaim 1 wherein: (a) said specimen is comprised of cytological materialthat is distributed as an adhering layer upon one surface of a slide;(b) said contacting is carried out by applying an aqueous solution ofsaid probe composition to said specimen using hybridizing conditions;(c) said separating is carried out by washing said resulting specimenwith an aqueous liquid; and (d) said examining is carried out with afluorescence microscope.
 16. The process of claim 1 wherein: (a) saidspecimen is comprised of an aqueous suspension containing said selectedchromosome or selected region; (b) said contacting is carried out bydissolving said probe composition in said suspension; (c) saidseparating is carried out by centrifuging; and (d) said examining iscarried out with a flow cytometer.